EFFECT OF Aspergillus flavus ON GROUNDNUT SEED QUALITY …oar.icrisat.org/9725/1/Adithya.pdf · groundnut resistant cv. J 11 4.6 Scanning electron micrographs of A. flavus growth

EFFECT OF Aspergillus flavus ON GROUNDNUT

SEED QUALITY AND ITS MANAGEMENT

GUNTHA ADITHYA

B. Sc. (Ag.)

THESIS SUBMITTED TO THE

PROFESSOR JAYASHANKAR TELANGANA STATE AGRICULTURAL

UNIVERSITY

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR

THE AWARD OF THE DEGREE OF

MASTER OF SCIENCE IN AGRICULTURE (SEED SCIENCE AND TECHNOLOGY)

CHAIRPERSON: Dr. B. RAJESWARI

DEPARTMENT OF SEED SCIENCE AND TECHNOLOGY

COLLEGE OF AGRICULTURE

RAJENDRANAGAR, HYDERABAD – 500 030


UNIVERSITY

2016

EFFECT OF Aspergillus flavus ON GROUNDNUT

SEED QUALITY AND ITS MANAGEMENT

GUNTHA ADITHYA

B. Sc. (Ag.)

THESIS SUBMITTED TO THE


UNIVERSITY

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR

THE AWARD OF THE DEGREE OF

MASTER OF SCIENCE IN AGRICULTURE (SEED SCIENCE AND TECHNOLOGY)

CHAIRPERSON: Dr. B. RAJESWARI

DEPARTMENT OF SEED SCIENCE AND TECHNOLOGY

COLLEGE OF AGRICULTURE

RAJENDRANAGAR, HYDERABAD – 500 030


UNIVERSITY

2016

DECLARATION

I, Mr. G. ADITHYA hereby declare that the thesis entitled “EFFECT OF Aspergillus

flavus ON GROUNDNUT SEED QUALITY AND ITS MANAGEMENT” submitted to the

Professor Jayashankar Telangana State Agricultural University for the degree of Master of Science

in Agriculture is the result of original research work done by me. I also declare that any material

contained in the thesis has not been published earlier in any manner.

Date: (G. ADITHYA)

Place: Hyderabad I. D. No. RAM/14-51

CERTIFICATE

Mr. G. ADITHYA has satisfactorily prosecuted the course of research and that the thesis

entitled “EFFECT OF Aspergillus flavus ON GROUNDNUT SEED QUALITY AND ITS

MANAGEMENT” submitted is the result of original research work and is of sufficiently high

standard to warrant its presentation to the examination. I also certify that neither the thesis nor its

part thereof has been previously submitted by him for a degree of any University.

Date: (Dr. B. RAJESWARI)

Place: Hyderabad Chairperson

CERTIFICATE

This is to certify that the thesis entitled “EFFECT OF Aspergillus flavus ON

GROUNDNUT SEED QUALITY AND ITS MANAGEMNT” submitted in partial fulfilment of

the requirements for the degree of Master of Science in Agriculture of the Professor Jayashankar

Telangana State Agricultural University, Hyderabad is a record of the bonafide original research

work carried out by Mr. G. ADITHYA under our guidance and supervision.

No part of the thesis has been submitted by the student for any other degree or diploma. The

published part and all assistance received during the course of the investigations have been duly

acknowledged by the author of the thesis.

CHAIRMAN

ADVISORY COMMITTEE

Thesis approved by the Student Advisory Committee

Chairperson: Dr. B. RAJESWARI

Senior Scientist, Department of Plant Pathology

RARS, Jagtial,

PJTSAU, Hyderabad.

----------------------------

Member: DR. K. KESHAVULU

Director

Telangana State Seed Certification Agency ----------------------------

Hyderabad, Telangana

Member: Dr. HARI KISHAN SUDINI

Senior Scientist

Groundnut Pathology, Grain Legumes ----------------------------

ICRISAT, Patancheru,

Hyderabad – 502 324

Date of final viva-voce:

ACKNOWLEDGEMENTS

First of all, I bow my head before my beloved parents ANANDAM and RENUKA for their

endless blessings and giving me patience, encouragement, strength, confidence, ability to achieve

this academic milestone. Knowledge in itself is a continuous process. I would have never succeeded

in completing my task with the cooperation, encouragement and help provided to me by various

personalities.

Firstly, with deep regards and profound respect, I avail this opportunity to express my deep

sense of gratitude and indebtedness to Dr. B. Rajeswari, Senior Scientist, Plant Pathology for her

inspiring guidance, constructive criticism and valuable suggestions throughout the research work.

It would have not been possible for me to bring out this thesis without her help and constant

encouragement. I feel proud of having a superb mentor in the scholarly person and it was really a

blessing for me to get to know her and work with her.

I deem it my privilege in expressing my deep sense of reverence and gratitude and

indebtedness to Dr. Hari Kishan Sudini, senior scientist, Groundnut Pathology, ICRISAT,

Patancheru and member of my advisory committee for her valuable suggestions, careful and

reasoned criticism and meticulous attention to the details and also for her constant encouragement

which has led to the present investigation to the final shape.

I owe my flow of thanks to Dr. K. Keshavulu, Director, Telangana State Seed Certification

Agency, Hyderabad, Telangana state and member of Advisory Committee for his worthful

suggestions and encouragement during the investigation.

I am very much thankful to and feel it a great privilege to place on my record with sincere

regards and thanks to Dr. K. Jhansi Rani, Associate Professor & Head, Dr. Razia Sultana,

Associate Professor, Dr. P. Sujatha, Assistant Professor, Department of Seed Science and

Technology, College of Agriculture, Rajendranagar, Hyderabad for their inspiring, insightful,

scholarly guidance and constructive suggestions from time to time in the preparation of this

dissertation.

I feel it a rare opportunity to express my bountiful regards, affection and gratitude to all the

teachers of my life Ragavendar reddy sir, Srinivas sir, Sudershan sir, Narsaiah sir, Aagaiah sir,

Sridar sir, Venumadav sir, Dr. Uma Maheshwari madam, Dr. Sridevi madam, Dr. Aruna kumari

madam, Dr. Radha Krishna sir, Dr. Manohar sir, Dr. Narayan reddy sir, Dr. Kishor verma sir,

Dr. Shilaja madam, Dr. Vani madam and who ever teaches me during my schooling, intermediate,

bachelors and masters degree who constantly inspired me to study and helped me in molding my

life.

The words are not enough to express all my love and thankfulness towards my family for

allowing me to pursue my research. It is because of their blessings that I could reach here. It gives

me a great pleasure to thank my grandmother Buchchamma, brothers Late Santosh, Aravind,

Shashidhar, Karthik, Nagarjuna and my beloved cousins Pavan, Pranav, Karunya, Ranjith,

Sandeep, Ravali, Sandya, Lavanya, Sahiti, Sindu, Kavya, Mamatha for their love,

encouragement, solid support and their understanding throughout my research work.

I express my thanks to my dear friends Raju, Sai, Raidu, Vijay Reddy, Vijay, Vinod, Appy,

Swathi madam, Harinayak, Joseph, Mukund, Dada, Chandu, Hari, Aravind, Naresh goud,

Deepak Reddy, Ajay, Ganesh, Sai Krishna, Mahendar, Chiranjeevi, Tirumal, Srinivasulu, Sashi,

Veerababu, Sathyanvesh, Mallesh, Nikil reddy, Navenn reddy, Ramesh, Prasana Krishna,

Nagaraju, Vishal, Anil, Prashanth, Raju, Ajay, Sathish, Sravan, Venkatesh, Uday, Naresh,

Narendar, Tarun, Mahesh, Karthik, Suresh, Yaswanth, Anushu, Mounika, Manasa, Madhuri

constant encouragement and ample support at all stages of my schooling, intermediate, bachelors

and masters degree who constantly inspired me to study and helped me in molding my life.

I thank all my seniors Suman, Srijan, Yellagoud, Sadaiah, Rajashekar, Satish,

Ramakrishna, Sai, Mahesh babu, Srinivas Reddy, Kranthi, Rakesh, Raju, Durgaraju,

Thiruchandaran who helped me in various ways.

I would like to acknowledge the constant assistance provided by my colleagues, Hari, Joseph,

Jagdeesh, Viran, Nagesh, Meena, Manjegowda and Sarita. I extended warmest thanks to my

seniors Santosh, Madan mohan reddy, Sowmya, Ramya, Asha Jyothi, Neelima and juniors

Prashanth and Sampath.

I am thankful to non-teaching staff of Department of Seed Science and Technology

Venkatesh, Krishnafer, Raghavendra, Nirmala for their help and care during my research work.

Special thanks to Mr. S. Veera Reddy, Dr. Naga Mangala, Dr. Srilaxmi, Smt. Rohini,

Mr.Noorulla haveri, Mr. Vijay Raju, Ms. Simi Jacob, Smt. Anasuyamma, Smt. Anitha Sri.

Ramachandraiah, Sri. Bhasker Raju, Sri. Prabhakar Reddy and Sri Ravinder Rao for being there

for me and making my stay at ICRISAT a memorable one.

I convey my whole hearted thanks to all my well wishers who were far too numerous to have

been mentioned here.

The financial assistance rendered by ICRISAT and PJTS Agricultural University in the

form of stipend is greatly acknowledged.

Place: Hyderabad

Date: GUNTHA ADITHYA

LIST OF CONTENTS

Chapter No. Title Page No,

I INTRODUCTION

II REVIEW OF LITERATURE

III MATERIAL AND METHODS

IV RESULTS AND DISCUSSION

V SUMMARY AND CONCLUSIONS

LITERATURE CITED

LIST OF PLATES

Plate No Title

3.1

Enzyme linked immunosorbent assay for estimation of aflatoxin

content in groundnut kernels

3.2

Pure culture of Pseudomonas fluorescens maintained on Kings B

medium

3.3 Pure culture of T. harzianum and T. viride maintained on PDA

4.1 Seed mycoflora detected by agar plate method (Farmer samples)

4.2 Seed mycoflora detected by agar plate method (Market samples)

4.3

External seed colonization of A. flavus in groundnut cv. J 11 at

different days of incubation period

4.4

External seed colonization of A. flavus in groundnut cv. JL 24 different

days of incubation period

4.5 Scanning electron micrographs of A. flavus growth in treated seeds of

groundnut resistant cv. J 11

4.6 Scanning electron micrographs of A. flavus growth in treated seeds of

groundnut susceptible cv. JL 24

4.7 Evaluation of seed treatments using bioagents and fungicides in

groundnut susceptible cv. JL 24 under glasshouse conditions

LIST OF TABLES

Table No TITLE

3.1 Collection of groundnut pod samples from different districts of

Telangana State

3.2 Effect of Aspergillus flavus infection on quality (oil, protein and fatty

acids) in groundnut cvs. J 11 and JL 24

4.1 Detection of seed mycoflora associated with groundnut farmer samples

collected from Karimnagar, Warangal, Nizamabad and Mahabubnagar

of Telangana state following agar plate method

4.2 Detection of seed mycoflora associated with groundnut market samples

collected from Karimnagar, Warangal, Nizamabad and Mahabubnagar

of Telangana state following agar plate method

4.3 Aspergillus flavus seed colonization severity scale on groundnut kernels

4.4 Aspergillus flavus seed colonization in groundnut cv. J 11 and JL 24 at

different days of incubation period

4.5 Effect of A. flavus infection on oil content (%) in treated and untreated

seeds of groundnut cvs. J 11 and JL 24

4.6 Effect of A. flavus infection on protein content (%) in treated and

untreated seeds of groundnut cvs. J 11 and JL 24

4.7 Effect of A. flavus infection on saturated fatty acids (palmitic acid)

content (%) in treated and untreated seeds of groundnut cvs. J 11 and JL

24

4.8 Effect of A. flavus infection on saturated fatty acids (stearic acid)


24

4.9 Effect of A. flavus infection on unsaturated fatty acids (linoleic acid)


24

4.10 Effect of A. flavus infection on unsaturated fatty acids (oleic acid)


24

4.11 Effect of A. flavus infection on aflatoxin content in treated and untreated

seeds of groundnut cvs. J 11 and JL 24 at different days of incubation

period

Table No TITLE

4.12 Evaluation of seed treatments with bioagents and fungicides against

A. flavus under glasshouse conditions

4.13 Estimation of aflatoxin content (µg/kg) in the harvested produce of

groundnut cv. JL 24 treated with bioagents and fungicides

LIST OF FIGURES

Figure No TITLE

4.1 Total seed mycoflora detected in groundnut farmer samples collected

from different districts of telangana state.

4.2 Total seed mycoflora detected in groundnut market samples collected

from different districts of telangana state.

4.3 Oil content in treated and untreated seed samples of groundnut cultivars

4.4 Protein content in treated and untreated seed samples of groundnut

cultivars

4.5 Palmitic acid content in treated and untreated seed samples of groundnut

cultivars

4.6 Stearic acid content in treated and untreated seed samples of groundnut

cultivars

4.7 Linoleic acid content acid in treated and untreated seed samples of

groundnut cultivars

4.8 Oleic acid content in treated and untreated seed samples of groundnut

cultivars

4.9

Effect of A. flavus infection on aflatoxin (µg kg-1) content in treated and

untreated seeds of groundnut cvs. J 11 and JL 24 at different days of

incubation period


A. flavus on germination


A. flavus on plant height


A. flavus on yield parameters

ABBREVATIONS

Af : Aspergillus flavus

CFU : Colony forming units

cm : Centimeter

CRD : Completely Randomized Design

ELISA : Enzyme- linked immunosorbent assay

et al. : Co-workers

etc : And so on

Fig : Figure

g : Gram

i.e., : That is

Kg : Kilogram

KMNO4 : Potassium permanganate

L : Litre

M : Molar

M : Million

mg : Milligrams

ml : Milliliter

mm : Millimeter

Mt : Million tones

N : Normal

NA : Nutrient Agar

ng : nano gram

nm : Nano metre

No. : Number 0C : Degree Centigrade

PDA : Potato Dextrose Agar

Pg : Pictogram

pH : Hydrogen ion concentration

Sp : Species

viz., : Namely

µg/Kg : Microgram per kilogram

% : Per cent

@ : At the rate of

µg : Microgram(s)

µL : Microlitre

CD : Critical difference

DAS : Days after sowing

Fig : Figure

ha : hectares

TFC : Total fungal colonies

S.Em (±) : Standard error of mean

min : minutes

h : hours

Author : G. ADITHYA

Title of the thesis : EFFECT OF Aspergillus flavus ON

GROUNDNUT SEED QUALITY AND ITS

MANAGEMENT

Degree : MASTER OF SCIENCE

Faculty :

AGRICULTURE

Discipline : SEED SCIENCE AND TECHNOLOGY

Major Advisor : Dr. B. RAJESWARI

University : PROFESSOR JAYASHANKAR

TELANGANA STATE AGRICULTURAL

UNIVERSITY

Year of submission

: 2016

ABSTRACT

Groundnut (Arachis hypogaea L.) is an important oil seed crop in India. It contains oil to an

extent of 48 - 51 %. The major problem associated with groundnut is aflatoxin contamination. It is

mainly caused by Aspergillus flavus and Aspergillus parasiticus. Groundnut being an oil seed, it

contains lesser amount of carbohydrates than cereals but more amount of oil and protein and they

break down into simple sugars and amino acids which is essential for germinating seed as an energy

source. Several management strategies were adopted to minimize the aflatoxin problem viz.,

development of resistant varieties, use of biocontrol agents and cultural practices. Keeping this in

view, the present findings pertaining to the present investigations were carried out on detection of

seed mycoflora, mode of entry of A. flavus into groundnut seed, effect of A. flavus on seed and oil

quality and evaluation of bioagents and fungicides in the management of A. flavus of groundnut

under glasshouse conditions.

A total of seventy two groundnut (72) pod samples comprising farmer samples (36) and

market samples (36) from major groundnut growing districts of Telangana state during 2015 - 2016.

The seed samples were analysed for seed health by agar plate method as per ISTA (1996).

Significant differences in occurrence of total number fungal colonies due to location and source of

seed samples were observed. Irrespective of the districts, total per cent occurrence of seed

mycoflora was found high in farmer samples (92.4 %) over market samples (45.3 %). Out of four

districts, samples of Mahabubnagar district (47.1 % & 26.2 %) followed by Warangal district (43.5

% & 24.6 %) recorded more total number of fungal colonies in farmer and market samples.

Irrespective of the samples, occurrence of six fungal flora viz., A. flavus, A. niger, Fusarium sp.

Alternaria sp. Macrophomina sp. Penicillium sp. were observed. Among them, A. flavus (43.2 %),

A. niger (26.7 %) were found predominant in both farmer and market samples.

External seed colonization due to A. flavus in groundnut resistant cv. J 11 and susceptible

cv. JL 24 were observed at different days of incubation period indicated that resistant groundnut cv.

J 11 inoculated with A. flavus colonized the seeds with severity score of 1, 2, 3, 4 and susceptible

cv. JL 24 inoculated with A. flavus colonized the seeds with disease severity score of 2, 3, 4, 4 at 3,

5, 7 and 9 days after incubation period.

The mode of entry of pathogen into groundnut seed was studied by Scanning Electron

Microscopy. Groundnut seeds of resistant and susceptible cultivars inoculated with A. flavus

http://www.scialert.net/asci/result.php?searchin=Keywords&cat=&ascicat=ALL&Submit=Search&keyword=amino+acid

toxigenic strain, the penetration and establishment of the fungi in case of J 11 was slow compared to

JL 24. The present investigation reveals that A. flavus is seed borne in nature and contaminated

seeds were important source of inoculum for seed infection and spread of the fungus from one seed

to another during storage.

The per cent reduction in oil content was high in susceptible groundnut cv. JL 24 (18.3%) as

compared to resistant groundnut cv. J 11 (9 %). While the reduction in oil content was less in the

untreated seeds of groundnut cv. JL 24 and groundnut cv. J 11 ( 13.7% and 6%). Overall the per

cent reduction in the protein content was found high in susceptible groundnut cv. JL 24 (16.3 %) as

compared to resistant groundnut cv. J 11 (6.5 %). While the reduction in protein content was less in

the untreated seeds of groundnut cvs. JL 24 and J 11 (14.2 % & 5.1 %).

The per cent reduction in the unsaturated fatty acids like linoleic and oleic acids were high

in susceptible groundnut cv. JL 24 (17.5 % and 16.6 %) as compared to resistant groundnut cv. J 11

(15 % and 14 %). Whereas in the untreated seeds, the per cent reduction in linoleic and oleic acids

were found low (11.3 % and 6 %) in cv. J 11 and 13.3 % and 8.2 % in cv. JL 24, respectively. The

increase levels of saturated fatty acids viz., palmitic and stearic acids were high in susceptible cv.

JL 24 (4.5 % & 4.5 %) as compared to resistant cv. J 11 (3.9 % & 2.93 %). Where as in untreated

seeds, the increased levels in palmitic and stearic acids were found low (2.5 & 2 %) in cv. J 11 and

2.9 % and 1.94 % in groundnut cv. JL 24 respectively.

The aflatoxin content at 1 to 56 days after incubation increased in groundnut cv. J 11 (2.15

µg/kg - 2861.3 µg/kg) & 63.4 µg/kg - 4077.1 µg/kg in cv. JL 24, respectively. In the untreated

seeds there was low level aflatoxin content of 2.15 µg/kg - 14.7 µg/kg in cv. J 11 and 2.15 µg/kg -

21.1 µg/kg in cv. JL 24 were recorded.

The efficacy of seed treatments against seed borne A. flavus were evaluated under glasshouse

conditions. Groundnut seeds treated with T. harzianum was significantly superior in recording

higher seed germination (96 %), plant height (4.75, 12.9 and 14.1 cm) and yield (4.60 g) followed

by T. viride (91 %, 4.10, 11.5 and 13.5 cm, 4.20 g) which was on par with P. fluorescens (88.2 %,

3.40, 10.2 and 10.8 cm 4.10 g). The remaining seed treatments were also found effective in

improving seed germination, plant height and yield in seeds treated with carbendazim (81 %, 2.77,

8.02 and 9.21 cm, 3.65 g), mancozeb (73.5 %, 2.72, 6.92 and 8.43 cm, 3.37 g) over untreated (65 %,

2.67, 6.62 and 7.37 cm, 29.2) and pathogen treated seeds (54.5 %, 2.57, 5.65, 6.41 cm, 2.55 g) at

15, 30, 45 DAS.

Aflatoxins were detected in pathogen treated seeds (1.38 µg/kg) and untreated seeds (0.69

µg/kg) which is below permissible level. While aflatoxin was not observed in the seed treated with

T. harzianum, T. viride and P. fluorescens.

CHAPTER I

INTRODUCTION

Groundnut (Arachis hypogaea L.) is one of the important oil seed crop grown all over the

world. India stands first in production and area under legumes in the world. The major crops include

groundnut, gram, pigeon pea, green gram and black gram. India is one of the largest producers of

oilseeds in the world and occupies an important position in the Indian agricultural economy. The

seeds of groundnut contains oil to an extent of 48 - 51 %. The crop is a rich source of protein,

dietary fiber, minerals and vitamins.

In India, groundnut is cultivated during kharif under rainfed conditions and in rabi and

summer seasons under irrigated conditions. 90 - 95 % of the total crop area is grown in kharif

season. It is cultivated in an area of 25.4 M ha worldwide with an annual production of 45.20 M t

and productivity of 3824 kg ha-1 (FAO, 2013). In India, the crop is grown to an extent of 5.53 M ha

with a production of 9.67 M t and productivity of 1750 kg ha-1 (INDIASTAT, 2013). In united

Andhra Pradesh, the crop is grown to an extent of 1.37 M ha with a production of 1.01 M t and

productivity of 890 kg ha-1. In Telangana state, the crop is grown to an extent of 0.21 M ha with a

production of 0.35 M t and productivity of 1690 kg ha-1 (Directorate of Economics and Statistics,

2015).

Availability of good quality seeds of high yielding varieties is the key to increase the

productivity. Groundnut production all over the world is limited by various biotic and abiotic

constraints that results in severe yield reduction. Seed borne pathogens affect the seed quality and

lower the yield. Knowledge on the type of the pathogen associated with farmers seed and their

effect on seed quality helps in adopting suitable strategies to manage them.

Deterioration in seed quality of groundnut is mainly due to A. flavus which makes the product

unfit for marketing and consumption. In groundnut, seed and seedling decay and aflarot diseases

were caused due to A. flavus pathogen. Aflatoxin contamination in groundnut kernels possesses a

great threat to humans and live stock health as well as international trade. According to FAO

estimates, 25 % of world food crops are affected by mycotoxins each year and also crop losses due

to aflatoxin contamination. Considering the significance of the aflatoxins, several countries

including FAO has fixed the tolerance limits for groundnut and its by-products. India and USA has

fixed the tolerance limit of 30 and 20 µg/kg of seed which is meant for human consumption

purpose. Aflatoxin contamination of agricultural crops such as groundnut and cereals causes annual

losses of more than $750 million in Africa. There are four major types of aflatoxins (namely

Aflatoxin B1, Aflatoxin B2, Aflatoxin G1 and Aflatoxin G2) among 18 structurally related

mycotoxins (Bennet, 2010). Aflatoxins designated by B1 and B2 shows strong blue fluorescence

under UV light, whereas G1 and G2 forms shows greenish yellow fluorescence.

In addition to this, environmental conditions also play a major role in the attack of these

molds and the crop is affected at various stages such as pre, post-harvest and storage conditions

(Waliyar et al., 2008). The extent of aflatoxin contamination in groundnut oil mills/traders gives an

indication of the prevalence of this qualitative problem. The pathogen produces aflatoxin as a

secondary metabolite in the seeds of number of crops both before and after harvest. It is a potent

carcinogen that is highly regulated in most of the countries.

Groundnut seeds are known to harbor several species of seed borne fungi viz., Aspergillus

flavus, Aspergillus niger, Macrophomina phaseolina, Rhizoctonia solani, Fusarium oxysporum, F.

solani were predominant in groundnut and seed coat was greatly infected by fungi followed by

cotyledon and axis (Rasheed et al., 2004). Species of Aspergillus, Penicillium, Fusarium, Rhizopus

and Alternaria are commonly occurring post harvest moulds in storage conditions (Chavan, 2011).

The major problems associated with groundnut is aflatoxin contamination. It is mainly caused by A.

flavus and A. parasiticus (Shephard, 2003 and Strosnider et al., 2006). The pathogen is saprophytic

soil fungus that infects and contaminates during pre and post-harvest stages of the groundnut crop.

Groundnut being an oil seed, it contains lesser amount of carbohydrates than cereals but

more amount of oil and protein and they break down into simple sugars and amino acids which is

essential for germinating seed as an energy source. Reduction in oil and protein content and

increased levels of free fatty acids were noticed in the stored kernels than in the pods due to

invasion of storage fungi (Ramamoorthy and Karivaratharaju, 1989). Presently, there are no tools

that would measure the total oil content of groundnut seeds, economically and non-destructively.

Groundnut pods have to be shelled and cleaned before oil content is measured which takes time and

resources. Near-infrared reflectance spectroscopy (NIRS) is effectively utilised for analysis of

chemical and physical properties without sample preparation and applied for the analysis of quality

characteristics in food and agricultural commodities (Batten, 1998; Williams & Norris, 2001). The

effect of A. flavus on seed quality i.e., aflatoxin levels in the groundnut seed samples were estimated

by using an indirect competitive ELISA method (Enzyme-Linked Immuno Sorbent Assay).

Ultrastructural studies in understanding the mode of entry of A. flavus pathogen in groundnut

resistant and susceptible cultivars through scanning electron microscopy was useful to identify the

fungal structures and facilities correct diagnosis and detailed examination of taxonomic characters

of seed borne fungi.

http://www.scialert.net/asci/result.php?searchin=Keywords&cat=&ascicat=ALL&Submit=Search&keyword=amino+acid

http://scialert.net/fulltext/?doi=ppj.2013.127.134#1203524_ja

For successful production of any crop the seed must be sound and free from seed mycoflora

which interfere with seed germination and subsequent emergence of the crop. Seed treatment with

bioagents and fungicides is an economical and viable approach to protect seed and seedlings from

attack of the pathogens. Several management strategies were adopted to minimize the aflatoxin

problem viz., development of resistant varieties, use of biocontrol agents and cultural practices.

However, genetic resistance is not available in the cultivable groundnut germplasm. Hence, to

control aflatoxin problem in groundnut biological control is considered as one of the viable options.

Fluorescent Pseudomonads as bacterial biocontrol agents were effectively utilised in reducing pre-

harvest aflatoxin contamination. During root colonization, these bacteria produce antifungal

antibiotics and elicit induced systemic resistance in the host plant or interfere specifically with

fungal pathogenicity factors. Several isolates of Trichoderma and Pseudomonas were characterized

for their antagonism against A. flavus and for their biocontrol potential (Anjaiah and Thakur, 2000

and Desai et al., 2000).

Keeping this in view, the present study has been proposed with the following objectives.

1. To study the extent of seed mycoflora contamination in groundnut kernels at farmers and

traders level.

2. To study the effect of A. flavus infection on seed and oil quality.

3. To study the effect of seed treatments under glasshouse conditions against A. flavus in

groundnut.

CHAPTER II

REVIEW OF LITERATURE

The available literature on Effect of Aspergillus flavus on groundnut seed quality and its

management has been reviewed in this chapter. As the available literature on these aspects is scanty,

the literature pertaining to other crops has also been reviewed under the following headings.

2.1 Effect of aflatoxins on human beings

2.2 Economic importance

2.3 Etiology of A. flavus

2.4 Seed mycoflora and its detection

2.5 Ultra structural studies

2.6 Effect of A. flavus on oil quality in groundnut

2.7 Estimation of oil and fatty acids by Near Infrared Reflectance Spectroscopy (NIRS)

2.8 Estimation of aflatoxins by Enzyme Linked Immunosorbent Assay (ELISA)

2.9 Management of A. flavus in groundnut using bioagents and fungicides under glasshouse

conditions

2.1 EFFECT OF AFLATOXINS ON HUMAN BEINGS

Aflatoxins are well recognized as a cause of liver cancer and other additional toxic effects.

In farm and laboratory animals chronic exposure to aflatoxins reduces immunity and interferes with

protein metabolism and multiple micronutrients that are critical to health. These effects have not

been studied in humans but the available information indicates that at least some of the effects

observed in animals were also observed in human beings.

Wild et al. (2002) reported the toxicity of aflatoxins by studying DNA adduct induction,

mutagenicity and carcinogenicity which is paralleled by the development of biomarkers of aflatoxin

exposure and biological effects which applied to human populations.

Agag et al. (2004) reported that aflatoxins affect the liver in which cytochrome P450

enzyme convert aflatoxins and capable of binding to both DNA and proteins. Inactivation of the p53

tumor suppressor gene leads to the development of liver cancer.

Crop storage conditions were frequently conducive for fungal growth and mycotoxin

production which effect on human health. The study also reveals that Aflatoxin B1 has been linked

to liver cancer such as immune suppression and growth faltering (Shepard, 2008).

Liu et al. (2010) conducted a study of quantitative cancer risk assessment by collecting

global data on food-borne aflatoxin levels, consumption of aflatoxin-contaminated foods and

hepatitis B virus (HBV) prevalence. It indicates that cancer potency of aflatoxin for HBV-positive

and HBV-negative individuals as well as uncertainty in all variables to estimate the global burden

of aflatoxin - related hepatocellular carcinoma (HCC).

Zain (2011) studied the impact of different categories of mycotoxin like aflatoxins,

ochratoxins, trichothecenes, zearalenone, fumonisins, tremorgenic toxins and ergot alkaloids on

human health. Further they reported that different factors which influence the presence of

mycotoxins in food were related to storage and other extrinsic factors such as climate or intrinsic

factors such as fungal strain specificity, strain variation and instability of toxigenic properties.

Eva et al. (2007) studied the impact of aflatoxins which causes different types of diseases

and disorders in human beings and animals.

Ding et al. (2012) reported the effect of aflatoxins on human dietary risk and their effects on

children by studying the 1040 groundnut seed samples collected from China after post harvest of

the crop.

2.2 ECONOMIC IMPORTANCE

The economic importance of aflatoxins derive directly from crop and livestock losses as

well as indirectly from the cost of regulatory programs designed to reduce risks to animal and

human health. Aflatoxin losses to livestock and poultry producers from aflatoxin contaminated

feeds include death and more effects on immune system suppression, reduced growth rates and

losses in feed efficiency. Other adverse economic effects of aflatoxins were include lower yields for

food and fiber crops.

The ability of aflatoxins to induce cancer and related diseases in humans indicates their

unavoidable occurrence in food and feeds make the prevention and detoxification of mycotoxins is

one of the most challenging toxicology issues of present time.

Health risks associated with aflatoxin consumption decreases labour productivity besides

increasing health costs and overall income losses due to opportunity costs linked to lost days of

work (Lubulwa and Davis, 1994).

According to Food and Agricultural Organization (FAO), 25 % of the world food crops are

significantly contaminated with mycotoxins (Boutrif and Canet, 1998). It is estimated that

approximately $ 450 million annual loss to all food exporters if all nations harmonized to EU

aflatoxin standards (Wu, 2004). Further, $ 670 million annual loss was incurred to African food

exporters from attempting to meet EU aflatoxin standards (Otsuki et al., 2001).

Aflatoxins were considered to have significant negative impact on health, food and

nutritional security and income at the household, community and national levels (Coulibaly et al.,

2008).

Mathur et al. (2015) reported that biotechnological tools to reduce aflatoxin contamination

through A. flavus by studying physical, biological and chemical methods.

2.3 ETIOLOGY OF A. flavus

Moreno et al. (1999) reported that A. flavus is propagated through conidial germination and

by hyphal growth. Sclerotial formations on maize kernels naturally infected by A. flavus and buried

sclerotia were able to withstand cold temperatures in the soil.

Afjal et al. (2013) studied Aspergillus species using macroscopic characteristics such as

colony growth, conidial color, colony reverse and microscopic characteristics including

conidiophore, vesicle, matulae, phialides and conidia for its identification.

Ihenacho et al. (2014) studied the morphological and molecular features of A. flavus and A.

parasiticus with the help of electrophoretic technique by studying DNA patterns.

2.4 SEED MYCOFLORA AND ITS DETECTION

Kishore et al. (2002) recorded the presence of mycotoxins and toxigenic fungi in 182

groundnut samples collected from farmers fields in five districts of Andhra Pradesh i.e., Anantapur,

Chittoor, Cuddapah, Kurnool and Mahabubnagar districts during rainy seasons of 1999 and 2000.

The seed infection due to A. flavus range was 11.9 % to 18.3 % and 9.5 % to 14.1 % in 1999 and

2000, respectively were recorded in each district.

Rasheed et al. (2004) studied the seed mycoflora of 12 groundnut seed samples by blotter,

agar plate and deep freeze method. Of the 14 genera, 28 species of fungi were isolated. All the

groundnut seed samples were infected by A. flavus where as 17 % with R. solani and Alternaria

citri, 33 % with M. phaseolina and 58 % with Fusarium sp. Aspergillus sp., as detected by blotter

method.

Nakai et al. (2008) reported the occurrence of seed mycoflora in stored groundnut samples

in Brazil. The results showed that predominance of Fusarium sp. (67.7 % in hulls and 25.8 % in

kernels) and Aspergillus sp. (10.3 % in hulls and 21.8 % in kernels) and the presence of five other

fungal genera. The toxigenic potential revealed that 93.8 % of the A. flavus strains isolated were

producers of AFB1 and AFB2 toxins.

Ibiam et al. (2011) evaluated fresh, cooked and fried seeds of three varieties of groundnuts

(Arachis hypogaea L.) Nwakara, Kaki and Campalla were screened to determine the post harvest

seed borne fungi. Fungi were not observed in fresh seeds of Nwakara and kaki varieties, where as

A.niger, A. flavus, A. terreus, A. culmorum, A. fumigatus, A. nidulans, A. tamarii, F. moniliforme,

Mucorrouxii, Penicillium spp, Cladosporium spp and Aureobasidium pullulans were found

associated with fried and cooked seeds of the three groundnut varieties Nwakara, Kaki and

Campalla.

Rathod et al. (2012) assessed the seed mycoflora of different varieties of legumes by

standard blotter paper, agar plate and seed wash methods. The results showed that agar plate

method was found effective with less incubation time and recording high percentage of seed

mycoflora.

Naqvi et al. (2013) studied seed quality viz., seed germination percentage, per cent pathogen

frequency and major seed-borne fungi of 30 groundnut seed samples. Fungi most frequently

isolated in groundnut seed were Alternaria, Aspergillus, Fusarium, Helminthosprium and Rhizopus.

The per cent pathogen frequency of seed-borne fungi was found high in groundnut (73.0 %).

Mohammed and Chala (2014) collected 270 groundnut samples from three districts of

Eastern Ethiopia and the incidence of infected groundnut kernels ranged from 50 to 80 % at the

district level. Heavy infestation of groundnut samples by various molds including A. niger, A.

flavus, A. ochraceus, A. parasiticus and Penicillium species were recorded with the kernel infection

varied kernel infection of 36.3 and 100 %.

Mohammed et al. (2015) studied 270 groundnut samples from farmers and storage fields

and local markets of three districts of Eastern Ethiopia for mycological analysis. A. flavus and A.

niger were isolated in higher frequencies from samples collected from farmers fields and stores than

markets while A. parasiticus was consistently isolated at higher frequencies than market samples.

At the district level, the incidence of infected groundnut kernels was ranged from 50 - 80 %.

Ramannuj et al. (2014) evaluated seed mycoflora of soybean, sorghum and groundnut in 18

villages of different zones of Madhyapradesh. Seed germination percentage, per cent pathogen

frequency and major seed-borne fungi were identified using blotter method. The per cent pathogen

frequency of seed-borne fungi was high in groundnut 73.0 % and low in soybean 15.3 %.

Nagpurne et al. (2014) conducted survey from Udgir region to detect seed mycoflora of five

different varieties of groundnut using blotter paper and agar plate methods. Seven genera of 12

species of fungi were isolated from this method. From the above study higher number of fungi

Aspergilli was isolated by blotter paper method (80 %) as compared to agar plate method (70 %).

Gintling et al. (2015) studied the physical quality and infection levels by collecting 16

groundnut samples from farmers, collectors, retailers and food processors in Banjarnegara District,

Central Java. On an average, the moisture content of groundnut kernels was 8.8 %, while the

damaged kernels (46.7 %) and A. flavus infection (45.1 %) were considerably high. From the above

study high damaged kernels and infection of A. flavus need to be decreased through proper handling

and storage practices.

2.5 ULTRASTRUCTURAL STUDIES

Mohan et al. (2003) screened 13 confectionary groundnut genotypes against A. flavus

seed colonization. The results revealed that cultivated groundnut genotypes showed stable

resistance to A. flavus and certain degree of resistance to seed colonization.

Ultra-structural studies using scanning electron microscopy for characterization of A. flavus

on sugarcane was found that conidia had two distinct ornamentations. The results confirmed that

conidia of A. flavus have relatively thin walls which were finely to moderately roughened. Further,

the conidial shape varied from spherical to elliptical (Rodrigues et al. 2007).

Achar et al. (2009) observed A. flavus infected groundnut kernels using light microscopy in

combination with electron microscopy to describe the infection course established by the pathogen.

Alvis et al. (2012) studied the SEM methodology to observe the interaction of fungi on seed

surface of cotton, common bean, soybean and maize. It has more advantage as compared to genome

techniques by considering the small structures of the fungi were measured and quantified by the

images stored in the computer and the results were analyzed in comparison with the conventional

methods.

2.6 EFFECT OF A. flavus ON OIL QUALITY IN GROUNDNUT

Deshpande et al. (1979) studied the colonization and biochemical changes in groundnut

seeds infected with A. flavus. The results showed that growth of A. flavus on various groundnut seed

samples resulted an increased levels of free fatty acids and decreased levels of protein content.

Deteriorative changes in oilseeds due to Aspergillus sp. recorded loss in protein content due

to A. terreus. It was found that fat content in groundnut and soybean was reduced due to A. flavus

(Chavan et al. 2003).

Maharous (2007) studied the chemical properties of A. flavus infected seeds exposed to

different levels of γ-irradiation during storage. The results revealed that there was no effect of

irradiation at different dose levels on moisture, protein, total lipids and amino acid content of the

seeds over a period of 60 days of seed storage.

Fagbohun et al. (2012) reported the nutritional and mycoflora changes in groundnut during

twenty weeks of storage period. A total of seven fungal species viz., A. flavus, A. niger, A.

fumigatus, Rhizopus sp., Penicillium sp., Mucor sp. and Fusarium sp. were recorded. Fungal

colonization and contamination of stored groundnut were found to reduce the market value,

edibility and depletion of nutrients.

Ameer et al. (2013) reported the relationship between seed borne pathogens and seed quality

deterioration of stored groundnut. The study reveals that a progressive decrease in germination

percentage, oil and protein content and increase in free fatty acid content in stored groundnut

kernels than pods were observed.

Begum et al. (2013) studied the relationship of A. flavus infection on seed quality by

artificial inoculation of A. flavus on seeds of groundnut cultivar VRI 2. Seeds with 0.25 % infection

maintained germination up to 71 % at the end of the storage period. Hence, this stage could be the

tolerable limit for the safe storage of groundnut seeds.

Bhushan et al. (2013) reported that high moisture content in the groundnut seeds resulted in

more fungal infection with Aspergillus sp. Aflatoxin contamination was higher at 18 % moisture

content as compared to other moisture regimes.

Adiver et al. (2015) conducted survey in 14 districts of Karnataka, India to assess the

severity of A. flavus. The results revealed that high incidence of A. flavus in the samples collected

from market areas. In inoculated groundnut seeds, the infection led to the reduction in sugars,

proteins, oil content and seed germination.

2.7 ESTIMATION OF OIL AND FATTY ACIDS BY NEAR INFRARED

REFLECTANCE SPECTROSCOPY (NIRS)

Ki won et al. (2000) studied 46 perilia and 83 groundnut samples to estimate lipid and

protein contents using NIRS equation development and validation and concluded that this method

was useful for mass screening of lipid and protein contents.

Sundaram et al. (2010) reported that groundnut oil and fatty acid concentration of Virginia

and Valencia types of in-shell groundnut using NIR reflectance spectroscopy. The average total oil

concentrations of all samples were determined by a standard soxtec extraction method and fatty

acids were converted to the corresponding methyl esters and measured using gas chromatography.

Patil et al. (2010) estimated fatty acid composition in 612 soybean seed samples using non

destructive method using Near Infrared Transmittance Spectroscopy. Highest variability was

observed for oleic and linoleic acid followed by palmitic and linolenic acid and least in stearic acid.

Sundaram et al. (2012) reported that moisture content of intact kernels of grain and nuts by

Near infrared reflectance spectrometry and In-shell groundnuts of two different market types

Virginia and Valencia were conditioned to different moisture levels between 6 % and 26 % and

separated into calibration and validation groups.

Rehema et al. (2014) assessed groundnut fatty acids by using Hyper Spectral Imaging (HSI)

method and it was an efficient and effective method for evaluating the quality and safety of oil.

Bansod et al. (2015) used non-destructive method to identify high oleic groundnut seeds to

support the selection and cultivation of high oleic acid groundnut varieties through NIRS.

2.8 ESTIMATION OF AFLAOXINS BY ENZYME LINKED

IMMUNOSORBENT ASSAY (ELISA)

Ramakrishna and Mehan (1993) studied direct and indirect competitive enzyme-linked

immunosorbent assays to determine the aflatoxin B1 in groundnut. For direct competitive assay, the

monoclonal antibody was conjugated to horse radish peroxidase (HRP) and for indirect competitive

ELISA a commercially available goat-antimouse Ig G-HRP conjugate was employed. The

sensitivities of both the ELISAs were as low as 20 pg/well and useful for routine analysis of

aflatoxin B1 in groundnut.

Aldao et al. (1995) quantified aflatoxin B1 by an indirect ELISA in groundnut samples and

observed the cross reactivity of antibodies with aflatoxin B2, G1 and G2.

Sylos et al. (1996) estimated aflatoxin content in 10 samples of groundnut and nine samples

of maize by ELISA and mini column chromatography and detected >20 μg/kg toxin in 50 per cent

groundnut seeds and none in maize samples and also ELISA method took less time to complete than

mini column chromatography.

Holbrook et al. (2000) tested 20 groundnut genotypes having drought tolerance and

susceptibility. The results showed that susceptible groundnut genotypes had greater pre harvest

aflatoxin contamination and drought tolerant genotypes had less pre harvest aflatoxin

contamination.

Reddy et al. (2001) collected three grades of chilli pod samples from the principal market

yards and cold storage facilities of the major chilli-growing areas of Andhra Pradesh (AP), India

(grades 1 to 3) in a survey during 1998 and 1999. An indirect competitive ELISA was used for the

estimation of aflatoxin B1 (AFB1) content. The results of indirect competitive ELISA revealed that

maximum per cent of grade 3 chilli pods have shown AFB1 levels higher than 30 μg kg-1 (non-

permissible levels) and one sample from grade 3 has highest AFB1 concentration (969 μg kg-1).

Asis et al. (2002) reported that aflatoxin B1 was highly contaminated groundnut samples

using HPLC and ELISA.

Kolosoya et al. (2006) developed direct competitive ELISA based on monoclonal antibody

and optimized for detection of aflatoxin and ELISA kit has been designed.

Lia Qi et al. (2006) detected aflatoxin B1 by ELISA and thin layer chromatography.

Radoi et al. (2008) developed different clones of antibodies against aflatoxin and their

efficacy was investigated by an indirect ELISA method.

Giray et al. (2009) used ELISA technique for the quantification of aflatoxin and ochratoxin

from 47 maize samples.

Li et al. (2009) reported that development of class specific monoclonal antibody based

ELISA for aflatoxin in groundnut.

Hong et al. (2010) studied the determination of aflatoxin B1 and B2 in groundnut and corn

based products by collecting 20 groundnut samples and corn based products from retail shop and

local market.

Ayejuyo et al. (2011) studied the assessment of aflatoxin levels in 99 samples of groundnut

through ELISA in Nigeria and among these 50 were contaminated with aflatoxin (50.5 %

incidence). The results showed that aflatoxin content of groundnut ranged from 6.25 ng/g to 7.80

ng/g.

Bakhiet and Musa (2011) analyzed sixty samples of stored groundnut kernels collected from

four different locations in Sudan were examined for aflatoxin contamination. Among these, thirty

five samples (58.3 %) were found positive with TLC technique and A. flavus was isolated from

twenty six samples (43.3 %). The concentration of aflatoxin B1 in these samples was ranged from

low (17.5 μg kg-1 kernel) to very high (404 μg kg-1 kernel).

Aseefa et al. (2012) studied the natural occurrence of toxigenic fungal species and aflatoxins

in freshly harvested groundnut kernels in Northern Ethiopia to detect the occurrence and severity of

infection. A total of 168 groundnut kernel samples were collected from farmers and research center

fields which recorded the aflatoxin concentrations with a range of 0.1 to 397.8 ppb.

Alemayechu et al. (2013) evaluated 120 samples from Ethiopia to assess the total aflatoxin

concentration in groundnut samples using an ELISA test. Of these, 93 were found positive while the

remaining 27 were found negative with aflatoxin level ranged from 15 mg kg-1 and 11,900 mg kg-1,

respectively.

Chala et al. (2013) studied 120 groundnut seed samples from farmers stores and markets in

Eastern Ethiopia to assess the natural occurrence of aflatoxins in surveyed samples through ELISA

method. Of these, 93 were found positive while the remaining 27 samples were found negative. The

total aflatoxin levels in the positive samples varied between 15 mg kg-1 and 11,900 mg kg-1.

Chen et al. (2013) reported that 1827 commercial groundnut products were analyzed for

aflatoxin levels which revealed that 32.7 % of samples with aflatoxin levels ranging from 0.2 mg

kg-1 to 513.4 mg kg-1. Aflatoxin B1 recorded the highest frequency of detection followed by

aflatoxin B2, aflatoxin G2 and aflatoxin G1.

Raarajan et al. (2013) assessed aflatoxin levels in groundnut seed samples collected from

trader godowns which were stored for several months. The study revealed that aflatoxin B1 played a

major role to study aflatoxin levels.

Khoraggani et al. (2013) evaluated the occurrence of aflatoxin in the groundnut samples

collected from supermarkets of Ahvaz were analyzed for the determination of aflatoxin

concentration in groundnut using TLC scanner. In total, 59.26 % of samples were contaminated

with aflatoxin, 14.8 % of samples were contain above 20 ppb which was above the maximum level

of total aflatoxin permitted in Iran.

Mohammed et al. (2015) studied different methods of mycological, biochemical and

molecular methods for detection of aflatoxigenic Aspergilli from groundnut kernels using PCR and

HPLC methods.

Rahmiana et al. (2015) studied the tolerant mechanism of different groundnut varieties

collected from Indonesia. These genotypes consisted of two local varieties, three drought tolerant

lines, one foliar disease tolerant line and four national-improved varieties. Aflatoxin content in

groundnut kernels varied among these Indonesian genotypes. The highest level of aflatoxin content

was in Tuban cultivar while the lowest was in GH 51 with 21 and 5 ppb, respectively.

Rahmiana et al. (2015) reported that factors leading to the post-harvest build up of aflatoxin

in groundnut sold in traditional market and in supermarket in Indonesia to assess the seed moisture

content, physical quality, A. flavus infection and aflatoxin B1 contamination. The results revealed

that seed water content at wholesalers, collectors and retailers in traditional wet markets was almost

lower than 10 %.

Waliyar et al. (2015) assessed aflatoxin contamination in three districts in Mali both in the

field and storage. Ninety groundnut pod samples in each district were collected from fields (30

villages/district and 3 samples/village) during 2009 and 2010. Pre-harvest contamination was

estimated at harvest whereas samples for post-harvest contamination were collected from granaries

of the same farmers at a monthly interval for 3 months. The results indicated that Mali groundnuts

are heavily contaminated with AFB1 at both pre and post-harvest stages and pose a serious threat to

human and animal health.

Wu et al. (2016) studied the aflatoxin contamination of groundnut at harvest in China from

2010 to 2013 and its relationship with climatic conditions by collecting 2494 groundnut samples.

The highest aflatoxin contamination level occurred under the climatic conditions where

precipitation was rare and the mean temperature was close to 230C and the minimum temperature

was approximately 200C and the maximum temperature was 290C.

2.9 MANAGEMENT OF A. flavus IN GROUNDNUT USING BIOAGENTS

AND FUNGICIDES UNDER GLASSHOUSE CONDITIONS

Mixon and Rogers (1973) suggested that use of groundnut cultivars resistant to seed

invasion and colonization by the aflatoxin producing fungi could be an effective means of

preventing aflatoxin contamination. They developed laboratory inoculation method for screening

groundnut genotypes resistance to A. flavus / A. parasiticus invasion and colonization.

Mixon et al. (1984) reported that chemical CGA 64250 and T. harzianum were found more

effective in reducing seed colonization by A. flavus in groundnut in the gypsum treated than in the

gypsum-untreated soils. There was no aflatoxin contamination of seeds in the gypsum-treated soil,

but it was found in seeds from the non-treated controls.

Mehan et al. (1987) reported resistance groundnut to seed infection of eleven genotypes

against A. flavus in field trials conducted in India. The results showed that positive correlations

were found between seed infection and aflatoxin contamination.

Waliyar et al. (1994) tested 25 groundnut lines for resistance to A. flavus colonization and

aflatoxin contamination. Average seed infection due to A. flavus varied with site and year from 5 %

to 37 % in groundnut cultivars viz., 55-437, J 11, and p1 337394 F were the least infected.

Romero et al. (2000) reported the efficacy of P. fluorescens isolates against A. flavus to

determine the inhibition of fungal infection on maize ear grains at the milk stage. It was observed

that mold infection was significantly reduced from 35.8 to 3.2 % on maize ears. Further, reduction

in mold infection was proportionate with an increased bacterial antagonist concentration.

Anjaiah et al. (2001) tested the efficacy of biocontrol strains potentially antagonistic to A.

flavus under in vitro conditions by dual culture plate method and concluded that 4 - 70 Trichoderma

isolates were found antagonistic.

Vijay et al. (2002) reported that predominance of A. fIavus infection in plot with farmers

practice (10 %) over improved package (2 %). It was due to inhibition of initial rhizosphere soil

population build up of Aspergillus by seed treatment with systemic fungicide and application of

biocontrol agent in the improved package.

Dharmaputra et al. (2003) studied the effect of non-toxigenic A. flavus, A.niger and T.

harzianum inoculated into planting media against toxigenic A. flavus infection and its aflatoxin

production in peanut kernels at harvest. Test fungi inoculated into planting media could inhibit

toxigenic A. flavus infection in groundnut kernels. Aflatoxin was detected in groundnut kernels

originated from one plant whose planting medium was inoculated only with the toxigenic A. flavus.

Thakur et al. (2003) reported that six Trichoderma and three Pseudomonas strains were

identified as highly antagonistic to Af 11-4 a highly toxigenic A. flavus strain in A. flavus-sick plots.

The antagonists were applied as seed dressing and soil application at flowering stage. Among the

biocontrol agents, two T. viride (Tv 17 and Tv 23), one T. harzianum (Th 23), and one

Pseudomonas (Pf 2) isolates provided greater protection to seed infection by Af 11 - 4.

Anjaiah et al. (2006) reported that inoculation of selected antagonistic strains fluorescent

Pseudomonads, Bacillus and Trichoderma sp. on groundnut have shown significant reduction of

seed infection by A. flavus. Further a reduction of >50 % of the A. flavus populations in the

geocarposphere of groundnut were observed.

Gachamo et al. (2008) studied the biocontrol management of aflatoxins using four

Trichoderma isolates under laboratory conditions. Two isolates of T. harzianum i.e., Th1, Th2 and

two isolates of T. viride i.e., Tv1 and Tv2 suppressed the growth of groundnut moulds and

significantly reduced the aflatoxins AFB1 and AFB2.

Reddy et al. (2009) reported that PGPR strains such as P. fluorescens and B. subtilis

inhibited A. flavus growth up to 93 % and 68 %, respectively. The fungal bioagents, T. virens

inhibited 80 % of the test pathogen in rice.

Palumbo et al. (2010) reported the efficacy of two bacterial strains, Pseudomonas

chlororaphis strain (JP1015) and P. fluorescens (strain JP2175) were tested in inhibiting the growth

of A. flavus under laboratory conditions. Further, three days after soil co-inoculation with P.

chlororaphis strain JP1015 inhibited A. flavus growth up to 100 fold and up to 58 fold by P.

fluorescens strain JP2175.

Rathod et al. (2010) studied the effect of fungicides on seed borne pathogen of groundnut.

The results indicated that thiram, carbendazim and mancozeb were found more inhibitory as

compared to other fungicides.

Verma et al. (2010) studied the interaction between antagonistic activity of P. fluorescens

on different sps. of Trichoderma. The inhibition in growth of Trichoderma was 8.59 % when

Trichoderma was used first and 59.4 % when P. fluorescens was used first in dual culture test.

Highest growth inhibition (59.4 %) of Trichoderma (Th3) was recorded when P. fluorescens was

first inoculated in King’s B medium 24 h prior to inoculation of T. harzianum. The population of

Trichoderma was gradually increased over a period of 14 days incubation.

Bhagawan et al. (2011) studied eco friendly management using five biocontrol agents like T.

viride, T. harzianum, T. hamatum, B. subtilis and P. fluorescens to reduce aflatoxin B1 in groundnut

cultivar GG - 20. The combination of T. viride, B. subtilis and P. fluorescens were found effective

in reducing A. flavus rhizospheric population, per cent incidence of afla root infection and

colonization of kernels and aflatoxin B1 content.

Baig et al. (2012) evaluated the efficacy of biocontrol agents, i.e., T. viride, T. harzianum, P.

fluorescens and B. subtilis against A. flavus, A. niger, F. oxysporum and A.alternata in oil seed

crops and found were effectively.

Dey et al. (2004) assessed nine different isolates of plant growth-promoting rhizobacteria

(PGPR) influence on plant growth, yield and nutrient uptake. Seed inoculation of these three

isolates, viz., PGPR1, PGPR2 and PGPR4 resulted significant increase in pod yield over control.

Pushpalatha et al. (2013) studied eco friendly management of seed borne fungi using

Trichococcus species in groundnut samples collected from various places of Karnataka. The fungal

pathogens were identified as Penicillium sp, A. niger, A. flavus and Fusarium sp. Among these,

maximum disease incidence due to A. niger was observed.

Sudha et al. (2013) evaluated efficacy of fungicides, bioagents and plant extracts against A.

flavus on chilli under both in vitro and field conditions. Among different bioagents tested P.

fluorescens and T. harzianum inhibited A. flavus growth up to 74 % and 70.4 % where as mancozeb

was effective with 91.1 % inhibition of A. flavus. Complete inhibition (100 %) of A. flavus was

recorded by the plant extracts like neem seed kernel extract (NSKE), nimbicidine and pongamia.

Sanskriti et al. (2015) reported the antifungal activity of P. fluorescens against A. flavus in

groundnut under laboratory and field trials. The results indicated that there was a significant

reduction in seed infection due to A. flavus by inoculation of P. fluorescens on groundnut.

Vasundara et al. (2015) studied the effect of seed treating fungicides and insecticides and

their combinations with T. viride on rhizosphere mycoflora and plant biometrics at 75 days after

sowing in groundnut.

CHAPTER III

MATERIAL AND METHODS

The present investigation was carried out at Department of Seed Science and Technology,

College of Agriculture, Rajendranagar, PJTSAU, Hyderabad, Telangana, in collaboration with

International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru,

Hyderabad, India. The details of the material and methods are presented here under the following

headings.

3.1 Collection of groundnut pod samples

3.2 Isolation of seed mycoflora by Agar plate method

3.3 Scanning electron microscopic studies (SEM)

3.4 Effect of A. flavus infection on oil quality

3.5 Evaluation of seed treatments (bioagents and fungicides) against A. flavus in

groundnut under glasshouse conditions

3.6 Estimation of seed quality (ELISA)

3.1 COLLECTION OF GROUNDNUT POD SAMPLES

A total of seventy two (72) pod samples were collected from the major groundnut growing

districts of Telangana viz., Warangal (18) Karimnagar (18), Nizamabad (18) and Mahabubnagar

(18) were collected. Out of 72 samples, 36 samples were collected from farmers and the remaining

36 samples were collected from market yards for assessment of seed mycoflora during 2015 - 2016.

The collected pod samples were shade dried and shelled and used for further studies.

Table 3.1 Collection of groundnut pod samples from different districts of Telangana State

S. No Name of the District

Surveyed

Source of sample

collection

(Farmer sample)

Source of sample

collection

(Market sample)

Number of

samples/district

1

Karmimnagar

Burampet Sultanabad Three

Sultanpur Choppadandi Three

Julapalli Husnabad Three

2

Warangal

Mulugu Hanmakonda Three

Keshavapatnam Warangal Three

Medaram Hasanparthi Three

3

Nizamabad

Balkonda Banswada Three

Bichkunda Nizamabad Three

Gandhari Bhodan Three

4

Mahabubnagar

Atmakur Narayankhade Three

Khanapur Jadcharla Three

Singampeta Nagarkurnool Three

Total number of samples collected 72

3.2 ISOLATION OF SEED MYCOFLORA BY AGAR PLATE METHOD

For isolation of seed mycoflora associated with groundnut samples, Agar plate method (ISTA,

1996) was employed.

3.2.1 Agar Plate Method (ISTA, 1996)

PDA medium was prepared by using the following components for isolation of the seed

mycoflora in the laboratory.

Potato dextrose agar (PDA)

Potato 200g

Dextrose 20g

Agar 20g

Water 1000 ml

PH 6.8

Peeled potato pieces were boiled in 500 ml of distilled water in a 1000 ml beaker till the

pieces got softened and the extract were collected in a beaker by sieving through a double layered

muslin cloth. Agar - agar (20g) was melted in another 500 ml of distilled water in 1000 ml beaker

into which 20g dextrose was added. The final volume of the medium was made up to 1000 ml by

adding sterile distilled water. The pH of the medium was adjusted to 6.8 with 0.1 N NaOH or 0.1 N

HCl as the case may be with the pH meter. The medium was sterilized in an autoclave at 15 psi for

15 minutes. About 20 ml of the medium was distributed to each of the sterile Petri plate under

aseptic conditions. Groundnut seeds were transferred to the plates containing PDA medium. Ten

seeds per plate were placed at equidistance in a circular fashion. Four hundred seeds from each

sample were placed in the plates in four replications. The Petri plates were incubated at 25 ± 20C in

an incubator for seven days and observed every day for the growth of fungi. Small quantity of

streptomycin sulphate was added in each plate for the suppression of the bacterial pathogens. The

characteristic features of the isolated seed borne fungi were tallied with the descriptions given for

identification (Ellis, 1976). The total fungal colonies were calculated and per cent infection was

assessed.

No. of seeds colonized in each plate by a

Particular species

Total fungal colonies (%) = ______________________________________________________ x 100

Total number of seeds in each plate

3.3 EXTERNAL SEED COLONIZATION BY A. flavus IN GROUNDNUT cvs.

J 11 AND JL 24

Seeds of groundnut cvs. J 11 and JL 24 were artificially inoculated with toxigenic strains of

A. flavus (isolate of Af 11 - 4) @ 109 conidia/ml were placed on sterilized petri plates and incubated

at 1, 3, 5, 7 and 9 days. Seeds of both the cvs. J 11 and JL 24 were assessed for surface seed

colonization by pathogen as per the colonization severity rating scale (1 - 4) as given by Thakur et

al. (2000).

3.4 SCANNING ELECTRON MICROSCOPIC STUDIES

Groundnut seeds of cv. JL 24 (susceptible) and cv. J 11 (resistant) were artificially

inoculated with A. flavus @ 109 conidia/ml and placed on sterilized blotter papers and maintained at

25 ± 20 C in a BOD incubator. Further seed samples were prepared with an interval of 48 h i.e., 1, 3,

5, 7 and 9 days after incubation. Ultra-structural mycelial characters in the infected groundnut seeds

were analyzed through Scanning Electron Microscopy (SEM) RUSKA laboratory, College of

Veterinary Science, SPVNRTSUVAFS, Rajendranagar, Hyderabad, Telangana state.

Infected groundnut seeds were cut into sections measuring not more than 1-2 mm with a

razor blade. Healthy groundnut seeds were aseptically washed and sectioned similarly to serve as

control treatments. Samples were fixed in 2.5 % glutaraldehyde in 0.1M phosphate buffer (pH 7.2)

for 24 h at 40C and post fixed in 2 % aqueous osmium tetroxide for 4 h. Dehydrated in series of

graded alcohols and dried to critical point drying with CPD unit. The processed samples were

mounted over the stubs with double - sided carbo conductivity tape and a thin layer of gold coat

over the samples were done by using an automated sputter coater (Model – JEOL JFC-1600) for 3

min and observed under Scanning Electron Microscope (SEM-Model: JEOLJFC-1600) at required

magnifications as per the standard procedures.

The basic steps involved in SEM sample preparation was surface cleaning, stabilizing the

sample with a fixative, rinsing, dehydrating, drying, mounting the specimen on a metal holder and

coating the sample with a layer of material that is electrically conductive.

3.4. 1 Cleaning the Surface of the Specimen

Cleaning of the sample was done to remove the media components and was permanently

fixed to the specimen surface. The sample was rinsed three times for 10 min in 0.1 M phosphate

buffer (pH 7.2) at room temperature.

3.4.2. Stabilizing the Specimen

Stabilization was done with fixatives. Samples were fixed by immersing the specimen in 2.5

% glutaraldehyde (GA) solution prepared in 0.1 M phosphate buffer (pH 7.2) and incubated at 40C

for 24 h and post fixed in 2 % aqueous osmium tetroxide (OsO4) for 4 h. The use of post fixative

helps in improving the bulk conductivity of the specimen.

3.4.3 Rinsing the Specimen

After fixation samples were rinsed in 0.1 M phosphate buffer (pH 7.3) one time for 10 min

and then three times for 20 min at 40C in order to remove the excess fixative.

3.4.4 Dehydration and Drying

Specimens were dehydrated in the following series of alcohols and dried in ethyl alcohol

critical point drying (CPD) unit.

30 % Ethyl alcohol - 10 min







100 % Acetone - 20 min

100 % Acetone - 20 min

This process allows the water in the samples to be slowly exchanged through liquids with

lower surface tensions.

3.4.5 Mounting the Specimen

The processed specimens were mounted on holder and inserted into the scanning electron

microscope. Samples were mounted on metallic (aluminum) stubs using a double sided carbon

conductivity tape.

3.4.6 Coating the Specimen

Specimens were coated with 20 nm to 30 nm thin layer gold coat over the samples by using

an automated sputter coater (Model - JEOL JFC - 1600) for 3 minutes and scanned under Scanning

Electron Microscope (SEM – Model: JOEL - JSM 5600) at required magnifications as per the

standard procedure.

3.5 EFFECT OF A. flavus INFECTION ON OIL QUALITY

Toxin producing aflatoxigenic strain of A. flavus (Af 11–4) was grown on potato dextrose

agar (PDA) and plates were kept in BOD incubator for 7 days at 25 ± 20C. Seeds of groundnut cv.

JL 24 (susceptible) and cv. J11 (resistant) were surface sterilized using 0.01 % clorax solution for

one min. Seeds were washed in three times with sterile water and placed in dry blotter paper to

remove the excess moisture. Seeds of both the cultivars were artificially inoculated with A.

flavus @ 109 conidia/ml and kept on sterilized blotter paper discs of 9 cm diameter and moistened

with sterile distilled water. The excess water was drained off from the plates. Groundnut seeds (400

Nos) in four replications were placed equidistantly on sterile blotter paper and incubated at 25 ± 20C

for a period of two months with an interval of 3, 7, 14, 21, 28, 35, 42, 49 and 56 days. After

incubation at different intervals groundnut seeds of both the cultivars (infected and healthy seeds)

were assessed for oil quality by using near infrared reflectance spectroscopy (NIRS) with the

following treatments.

Table 3.2. Effect of A. flavus infection on oil quality (Oil, Protein and fatty acids) in

groundnut cvs. J 11 and JL 24

Treatments Groundnut cv. J11 ( R) Groundnut cv. JL 24 ( S)

T1

(0 Day)

(J 11 + Af 11-4) (JL 24 + Af 11-4)

T2

(3rd Day)

(J 11 + Af 11-4)

Control (Untreated)

(JL 24 + Af 11-4)

Control (Untreated)

T3

(7th Day)

(J 11 + Af 11-4)

Control (Untreated)

(JL 24 + Af 11-4)

Control (Untreated)

T4

(14th Day)

(J 11 + Af 11-4)

Control (Untreated)

(JL 24 + Af 11-4)

Control (Untreated)

T5

(21st Day)

(J 11 + AF 11-4)

Control (Untreated)

(JL 24 + Af 11-4)

Control (Untreated)

T6

(28th Day)

(J 11 + Af 11-4)

Control (Untreated)

(JL 24 + Af 11-4)

Control (Untreated)

T7

(35th Day)

(J 11 + Af 11-4)

Control (Untreated)

(JL 24 + Af 11-4)

Control (Untreated)

T8

(42nd Day)

(J 11 + Af 11-4)

Control (Untreated)

(JL 24 + Af 11-4)

Control (Untreated)

T9

(49th Day)

(J 11 + Af 11-4)

Control (Untreated)

(JL 24 + Af 11-4)

Control (Untreated)

T10

(56th Day)

(J 11 + Af 11-4)

Control (Untreated)

(JL 24 + Af 11-4)

Control (Untreated)

The per cent oil, protein and fatty acids (saturated and unsaturated) contents were estimated

by using NIRS (model XDS RCA, FOSS Analytical AB, Sweden, Denmark). Non-destructive

method of estimation was used in NIRS. Approximately 70 - 100 g of each intact groundnut sample

was kept in rectangular cup in the NIR machine and readings were taken at different days of

incubation.

3.6 ESTIMATION OF SEED QUALITY BY USING ELISA (ENZYME

LINKED IMMUNOSORBANT ASSAY)

3.6.1. Coating

ELISA plates were coated with 150 µl of AFB1 – BSA conjugate (1 µl of AFB1 – BSA in 10

ml of 0.2 % carbonate buffer).

Incubated for overnight in refrigerator or incubator at 370 C for one hour.

Washed the plate thrice with PBS – T 20 (phosphate buffer saline – tween for 3 min).

3.6. 2 Blocking

160 µl of 0.2 % BSA (Bovine serum albumin) was added and incubated at 370 C for one

hour.

Wash the plate thrice with PBS – T 20.

Dilution of antiserum in a ratio of 1:2000 in a test tube and incubated at 370 C.

3.6.3 Competition

AFB1 standards ranging from 0.1 to 50 ng/ml were prepared in groundnut extracts (diluted

to 10 %) not containing any aflatoxin. Healthy groundnut kernels (20g) free of aflatoxin were

powdered and extracted in 100 ml of 70 % methanol containing 0.5 % KCl. The extract was filtered

and diluted to 1:10 in PBST – BSA. This was used as a diluent for aflatoxin standards.

Simultaneously pure toxin (AFB1) was prepared by diluting with above prepared healthy

groundnut (HGN) extract in a test tube.

100 µl of AFB1 (50 ng/ml) was added to first two columns of first two rows.

100 µl of diluted HGN extract was added to remaining wells of first two rows.

The remaining wells were loaded with 90 µl of BSA + 10 µl of sample extract to be

analyzed.

50 µl of antiserum was loaded to each well of ELISA plate and kept in shaker for 10 min.

Incubated the plate for one hour at 370 C to facilitate reaction between toxin and antibody

and plate was washed thrice in PBS – T 20.

Plate 3.1. Enzyme linked immunosorbent assay for estimation of aflatoxin content in

groundnut kernels. A) Microplate reader B) 96-well ELISA plate prior to

experimentation C) ELISA plate after the reaction

3.6.4 Conjugation

150 µl of substrate buffer {PNPP (P – Nitro Phenyl Phosphate) in 10 % diethylene amine}

was added to each well.

Simultaneously substrate was added to top left corner well as blank.

Incubated at normal temperature in dark for colour development at 15 minutes interval.

Absorbance was measured at 405 nm in ELISA reader.

ELISA Plate Reader (Bio-Rad)

Micropipettes: 1 - 40 l, 40 - 200 l and 200 - 1000 l single channel pipettes, 40 – 200 l

multichannel pipettes (Finn pipette) were used.

ELISA plates: For high binding ‘NUNC – MaxisorpTMsurface’ plates were used.

Others

Polyclonal antibodies for total aflatoxins

Mortar and pestle, Muslin cloth, pH meter, incubator, Refrigerator

Aflatoxin B1 standard (Sigma A6636)

Aflatoxin B1 - BSA conjugate (Sigma A6655)

Bovine Serum Albumin (Sigma A6793)

Solutions

Carbonate buffer or coating buffer (pH 9.6)

Na2CO3 --1.59 g

NaHCO3 --2.93 g

Distilled water --1000 ml

Phosphate buffer saline (PBS) (pH 7.4)

Na2HPO4 -- 02.38 g

KH2PO4 -- 00.40 g

KCl -- 00.40 g

NaCl -- 16.00 g

Distilled water --2000 ml

Phosphate buffer saline Tween (PBS-T)

PBS --1000 ml

Tween - 20 -- 0.5 ml

Antibody buffer

PBS-T --100 ml

Polyvinyl Pyrrolidone (PVP) 40,000 MW --2.0 g

Bovine serum albumin --0.2 g

3.6.5 Preparation of groundnut seed extracts

Groundnut seed (100 g) was grinded into powder using a blender. The seed powder was

titrated in 70% methanol (v/v-70 ml absolute methanol in 30 ml distilled water) containing 0.5 %

KCl (proportion used in 100 ml for 20 g seed) in a blender until the seed powder was thoroughly

ground. The extract was transferred to a conical flask and shaken for 30 min at 300 rpm in the

mechanical shaker. The extract was filtered through Whatman No. 1 filter paper. To estimate lower

levels of AFB1 (<10 g Kg-1), prior to ELISA, a simple liquid cleanup and concentration (5:1)

procedure was adopted. Twenty ml of methanol extract, 10 ml of distilled water and 20 ml of

chloroform were mixed in a separating funnel and used for cleanup. After vigorous shaking for one

min, the lower chloroform layer was collected and evaporated to near desiccation in water bath at

60°C. To the residue, four ml of PBS - Tween containing 7 % methanol was added and used for

analysis by ELISA.

AFB1 - BSA conjugate was prepared in carbonate coating buffer at 100 ng/ml concentrations

and 170 l of the diluted AFB1 - BSA is dispensed to each well of ELISA plate. The plate was

incubated in a refrigerator overnight at 37° C for at least one and half-hour.

The plates were washed in three changes of PBS – Tween allowing 3 min gap between for

each wash (To inhibit non-specific binding of antibodies and thus give false positive reaction). BSA

(0.2 %) was prepared in PBS - Tween was added in 170 µl per each well of ELISA plate and

incubated at 37°C for 1h. The plates were washed in three changes of PBS - Tween allowing 3 min

between each wash.

3.6.6 Preparation of Aflatoxin B1 standards

Healthy groundnut seed extract was prepared as mentioned previously. Aflatoxin B1

standards (using 1:10 healthy groundnut seed extract) were diluted at concentrations ranging from

100 ng to 10 picogram in 100 l volume.

Using the values obtained for aflatoxin B1 standards a curve was drawn with the help of a

computer, taking aflatoxin concentrations on the X-axis and optical density values on the Y-axis.

The amount of aflatoxin present in the sample was calculated using the formula given below:

AFB1

(μg/kg) =

A X D X E

or

A X E

G C X G

A = AFB1 concentration in diluted or concentrated sample extract (ng/ml)

D = Time dilution with buffer

C = Time concentration after clean up

E = Extraction solvent volume used (ml)

G = Sample weight (g)

3.7 EVALUATION OF SEED TREATMENTS (BIOAGENTS AND FUNGICIDES)

AGAINST A. flavus IN GROUNDNUT UNDER GLASSHOUSE CONDITIONS.

3.7.1 Methodology

Seeds of groundnut susceptible cv. JL 24 were surface sterilized and artificially inoculated

with A. flavus @ 109 conidia / ml. After 24h, the seeds were again treated separately with T.

harzianum @ 10 g kg-1, T. viride @ 10 g kg-1, P. fluorescens @ 10 g kg-1, mancozeb @ 2.5 g kg-1

and carbendazim @ 2 g kg-1 along with untreated and pathogen treated seeds. Seeds after

imposition of treatments were sown in sterilized soil filled in earthen pots containing 5 kg soil @

five seeds per pot in replicated trial adopting CRD design under controlled glasshouse conditions.

Observations were recorded on germination percentage, plant height and yield. Aflatoxin levels in

the harvested produce were estimated through ELISA. The details of the experiment were provided

in Table 3.5.1.

3.7.2 Treatments

Design CRD

Replications 4

Treatments 5

T1 Trichoderma harzianum @ 10 g/kg

T2 Trichoderma viride @ 10 g/kg

T3 Pseudomonas flourescens @ 10 g/kg

T4 Mancozeb @ 2.5 g/kg

T5 Carbendazim @ 2 g/kg

T6 Inoculated control (Treated seeds)

T7 Uninoculated control (untreated seeds)

Note: Seeds were prior inoculated with A. flavus @ 109 conidia / ml before imposition of seed

treatments from T1 to T6 except T7 treatment.

3.7.3 Observations

3.7.3.1 Germination (%)

The number of seeds germinated in each treatment was counted on seventh day after

sowing. Four replications were maintained for each treatment.

3. 7.3.2 Plant height (cm)

The plant height was recorded in cm from the base of the plant at 15, 30 and 45 DAS.

Harvesting

The crop was harvested at maturity, threshed and sun-dried for 3 days.

3. 7.3.3 Yield (g)

Pod weight in grams was recorded in different treatments with replication wise.

Plate 3.2. Pure culture of Pseudomonas fluorescens maintained on Kings B medium

Plate 3.3. Pure culture of T. harzianum and T. viride maintained on PDA

3.7.3.4 Aflatoxin levels

Pods after harvest were manually shelled and seeds were assessed for aflatoxin by following

the indirect competitive ELISA (Reddy et al. 2001).

3.8 STATISTICAL ANALYSIS

The data obtained in various laboratory/glasshouse experiments were statistically analyzed by

using Completely Randomized Design (CRD) as suggested by Gomez and Gomez (1984). The data

pertaining to percentage were angular transformed wherever necessary.

CHAPTER IV

RESULTS AND DISCUSSION

The results of the experiment conducted in the present investigation are presented here

under the following headings.

4.1 DETECTION OF SEED MYCOFLORA ASSOCIATED WITH

GROUNDNUT SEED SAMPLES COLLECTED FROM FARMERS

A total of 36 groundnut pod samples were collected from four major groundnut growing

districts of Telangana state viz., Karimnagar (9), Warangal (9), Nizamabad (9) and Mahabubnagar

(9). The seed samples were analyzed for seed health as per ISTA, 1996 by agar plate method. Seed

mycoflora associated with groundnut seed samples were isolated and identified.

4.1.1 Agar plate method

Mycoflora associated with groundnut seed samples from farmers and market yards of

Karimnagar, Warangal, Nizamabad and Mahabubnagar districts were detected following the agar

plate method (Fig 4.1).

Significant differences in occurrence of seed mycoflora in different districts of Telangana

state were observed. The results indicated that irrespective of the location and sources, a total of six

fungal species belonging to five genera were detected from the seed samples tested. Six fungi viz.,

Aspergillus flavus, A. niger, Fusarium sp. Alternaria sp. Macrophomina sp. and Penicillium sp.

were observed. Total per cent occurrence of seed mycoflora in different districts viz., Karimnagar

(72.2 %), Warangal (82.1 %), Nizamabad (56.7 %) and Mahabubnagar (84.8 %), were observed

respectively with respect to the mean total fungal colonies. Seed samples collected from

Mahabubnagar district (84.8 %) and Warangal district (82.1 %) recorded more total number of

fungal colonies followed by Karimnagar (72.2 %) and Nizamabad districts (56.7 %) which was

lowest of all the districts (Table 4.1).

Out of six fungal species recorded, the occurrence of A. flavus was predominant in the seed

samples analyzed from all the four districts (43.2 %). The occurrence of A. flavus was highest in the

seed samples of Mahabubnagar district (47.1 %) followed by Warangal district (43.5 %). A. flavus

was the most predominant fungus followed by A. niger (14.7 % to 34.4 %). The occurrence of

pathogenic fungi viz., Alternaria sp. (0.67 % to 0.89 %), Fusarium sp. (0.78 % to 1.90 %),

Macrophomina sp. (0.67 % to 1 %), Penicillium sp. (0.67 % to 1 %) were observed in these

Table 4.1. Detection of seed mycoflora associated with groundnut farmer samples collected from Karimnagar, Warangal, Nizamabad and

Mahabubnagar of Telangana state following agar plate method

S.

No.

DISTRICTS

SURVEYED

A. flavus

(%)

A. niger

(%)

Fusarium sp.

(%)

Alternaria sp.

(%)

Macrophomina sp.

(%)

Penicillium sp.

(%)

TFC

(%)

1 Karimnagar 43.1

(41.0)

25.5

(30.3)

1.00

(5.98)

0.89

(5.70)

1.00

(5.97)

0.67

(5.25) 72.2

2 Warangal 43.5

(41.2)

32.2

(34.5)

1.90

(7.61)

0.67

(5.26)

0.78

(5.45)

1.00

(5.97) 82.1

3 Nizamabad 39.1

(38.6)

14.7

(21.4)

0.78

(5.51)

0.67

(5.33)

0.67

(5.25)

0.78

(5.51) 56.7

4 Mahabubnagar 47.1

(43.4)

34.4

(35.8)

0.78

(5.51)

0.89

(5.62)

0.67

(5.97)

0.89

(5.70) 84.8

Mean 43.2 26.7 1.10 0.78 0.78 0.84

SE(m) ± 2.11 1.69 1.08 0.91 1.05 0.96

CD at 5 % 6.18 4.95 3.15 2.65 3.06 2.80

Figures in parenthesis are angular transformed values. Each value is mean of four replications. TFC: Total fungal colonies

Fig 4.1. Total seed mycoflora detected in groundnut farmer samples collected from different districts of telangana state.

0

5

10

15

20

25

30

35

40

45

50

A. flavus A. niger Fusarium Alternaria Macrophomina Penicillium

(%)

Seed mycoflora

Karimnagar

Warangal

Nizamabad

Mahabubnagar

Plate 4.1. Seed mycoflora detected by agar plate method (Farmer samples)

M:Mahabubnagar, W:Warangal, K:Karimnagar, N:Nizamabad

M W

K N

samples. The differences in occurrence of seed mycoflora in groundnut seed samples collected from

different districts may be attributed to the variations in the moisture content of the seed and storage

environment (temperature, relative humidity and light) adopted by the farmers. Mycoflora

associated with seed may varied from place to place due to change in conditions prevailing during

seed development, harvesting and storage. Seed mycoflora was highest in the seed samples of

Mahabubnagar district (84.8 %), while it was least in Nizamabad district (56.7 %). Storage fungi

like A. flavus, A. niger and Penicillium sp. were found low in Nizamabad district with a range of

0.67 % to 39.1 %. The per cent occurrence of the individual fungi was ranged from 0.67 % to 47.1

%. Seed mycoflora viz., A. flavus, A. niger, Fusarium sp. Alternaria sp. Macrophomina sp. and

Penicillium sp. were recorded in the groundnut seed samples indicated their seed borne nature in

groundnut. There is need for reducing the mold growth and mycotoxin production by improving the

storage conditions.

The present findings corroborates the report of Rathod et al. (2012) who reported that agar

plate method was effective in detection of seed borne fungi in groundnut. Agar plate method

favours the growth of fungi and gives highest per cent incidence due to potato dextrose agar

contents. He further reported the highest occurrence of A. flavus and A. niger. Most of the fungal

species detected in the present study were reported earlier in groundnut by Goldblatt (1969), Reddy

and Rao (1980), Mukherjee et al. (1992), Lumpungu et al. (1989), Rasheed et al. (2004), Shazia et

al. (2004), Aseefa et al. (2012), Naqui et al. (2013) and Nagapurne and Patwari (2014).

4.2 DETECTION OF SEED MYCOFLORA ASSOCIATED WITH

GROUNDNUT SEED SAMPLES COLLECTED FROM

MARKETYARDS

Significant differences in occurrence of seed mycoflora in different districts of Telangana

state were observed. The results indicated that irrespective of the location and sources, six fungal

species belonging to five genera were detected from the seed samples tested. Aspergillus flavus, A.

niger, Fusarium sp. Alternaria sp. Macrophomina sp and Penicillium sp. Total per cent occurrence

of seed mycoflora in different districts were ranged from Karimnagar (41.7 %), Warangal (50.4 %),

Nizamabad (34.7 %) and Mahabubnagar (54.8 %), respectively with respect to the mean total

fungal colonies. Seed samples collected from Mahabubnagar district (54.8 %) and Warangal district

(50.4 %) recorded more total number of fungal colonies followed by Karimnagar district (41.7 %).

Least total number of fungal colonies were observed in Nizamabad district (34.7 %) (Table 4.2,

Plate4.2).

Table 4.2. Detection of seed mycoflora associated with groundnut market samples collected from Karimnagar, Warangal, Nizamabad and

Mahabubnagar of Telangana state following agar plate method

S.

No.

DISTRICTS

SURVEYED

A. flavus

(%)

A. niger

(%)

Fusarium sp.

(%)

Alternaria sp.

(%)

Macrophomina sp.

(%)

Penicillium sp.

(%)

TFC

(%)

1 Karimnagar 19.5

(22.7)

19.6

(26.2)

0.78

(5.53)

0.56

(5.06)

0.67

(5.25)

0.67

(5.26) 41.7

2 Warangal 24.6

(25.9)

21.9

(27.8)

0.78

(5.51)

0.78

(5.45)

1.00

(5.98)

1.33

(6.54) 50.4

3 Nizamabad 15.6

(21.6)

16.7

(23.9)

0.78

(5.45)

0.78

(5.51)

0.44

(5.25)

0.44

(4.79) 34.7

4 Mahabubnagar 26.2

(30.7)

25.0

(29.9)

1.44

(6.80)

0.78

(5.51)

0.67

(5.97)

0.67

(5.33) 54.8

Mean 21.5 20.8 0.94 0.72 0.69 0.78

SE(m) ± 1.37 1.52 1.22 0.99 0.98 1.00

CD at 5 % 4.00 4.45 3.58 2.89 2.73 2.93

Figures in parenthesis are angular transformed values. Each value is mean of four replications. TFC: Total fungal colonies

Fig 4.2. Total seed mycoflora detected in groundnut market samples collected from different districts of telangana state.

0

5

10

15

20

25

30

A. flavus A. niger Fusarium Alternaria Macrophomina Penicillium

(%)

Seed mycoflora

Karimnagar

Warangal

Nizamabad

Mahabubnagar

Plate 4.2. Seed mycoflora detected by agar plate method (Market samples)

M:Mahabubnagar, W:Warangal, K:Karimnagar, N:Nizamabad

W

K N

M

Out of six fungal species recorded, the occurrence of A. flavus was found predominant in the seed

samples analyzed from four districts (21.5 %). The occurrence of A. flavus was found highest in the

seed samples of Mahabubnagar district (26.2 %) and Warangal district (24.6 %) and was the most

predominant fungus followed by A. niger (16.7 % to 25 %). The occurrence of pathogenic fungi

like Alternaria sp. (0.56 % to 0.78 %), Fusarium sp. (0.78 % to 1.44 %), Macrophomina sp. (0.44

% to 1 %), Penicillium sp. (0.44 % to 1.33 %) were observed in the seed samples analyzed from

four districts, respectively. Seed mycoflora was found highest in the seed samples of Mahabubnagar

district (54.8 %), while it was least in Nizamabad district (34.7 %). Storage fungi like A. flavus, A.

niger and Penicillium sp. were low in Nizamabad district (0.44 % to 16.7%). The per cent incidence

of the individual fungi was ranged from 0.44 % to 26.2 %. Seed mycoflora viz., A. flavus, A. niger,

Fusarium sp. Alternaria sp. Macrophomina sp. and Penicillium sp. were recorded in the groundnut

seed samples collected from market yards indicated their seed borne nature.

The present findings are in conformity with the earlier findings of Pitt and Hocking (1997),

Guchi et al. (2014) and Kalyani et al. (2014) who reported that A. flavus and A. niger were more

predominant in the field and stored foods than in the markets which might be attributed to source of

seed samples, place of collection and location.

4.3 EXTERNAL SEED COLONIZATION BY Aspergillus flavus IN

GROUNDNUT cvs. J 11 AND JL 24

Significant differences in seed colonization were observed in groundnut seeds of resistant

cv. J 11 and susceptible cv. JL 24 which were artificially inoculated with A. flavus toxigenic strain

(Af 11 - 4) @ 109 conidia/ml and incubated for 1, 3, 5, 7 and 9 days along with untreated seeds were

externally examined for seed colonization. The percent surface area colonized due to A. flavus with

a severity score of 1, 2, 3 and 4 were observed at 3, 5, 7 and 9 days of incubation in resistant cv. J

11. Whereas, in the susceptible cv. JL 24 recorded severity score of 2, 3, 4 and 4 at 3, 5, 7 and 9

days of incubation period indicating the differences in seed colonization in resistant and susceptible

cultivars (Table 4.3, 4.4) (Plate 4.3,4.4).

Similar variation in seed colonization due to A. flavus pathogen in groundnut accessions was

reported earlier by Deshpande and Pancholi (1979) and Thakur et al. (2000) and Nakai et al. (2008)

recorded the susceptibility of groundnuts to colonization of A. flavus especially during storage.

Plate 4.3. External seed colonization of A. flavus in groundnut cv. J 11 at different days of

incubation period

Day 1

Day 9 Day 7

Day 3 Day 5

Control

Plate 4.4. External seed colonization of A. flavus in groundnut cv. JL 24 at different days of

incubation period

Control Day 1

Day 7

Day 3 Day 5

Day 9

4.4 SCANNING ELECTRON MICROSCOPIC STUDIES

Groundnut seeds of resistant cv. J 11 and susceptible cv. JL 24 were artificially inoculated

with A. flavus @ 109 conidia/ml and incubated at 1, 3, 5, 7 and 9 days along with untreated seeds.

Nature of seed colonization by A. flavus and entry of the pathogen into the groundnut seeds was

observed with Scanning Electron Microscopy (SEM). The results revealed that presence of

pathogen mycelium in the damaged seed coat with fractured discontinuous epidermis with loose

broken cell junctions between epidermal cells were observed. In addition to this rough,

discontinuous, disorganised parenchyma, broken and missing cell walls and almost with total

depletion of storage proteins were observed. Similarly, cells of healthy embryos of both cultures

were well organized, unruptured cell walls with minimal intercellular space and abundant storage

proteins. Hyphae penetrated into embryonic tissues and established intercellularly and

intracellularly leading to an overall depletion of storage proteins. Intense hyphal branching with

haustoria and abundant sporulation were observed in groundnut cv. JL 24 as compared to resistant

cv. J 11 (Plate 4.5 & 4.6).

Seed coat responsiveness is a major key factor in establishing the pathogen when infection

occurs. In the present study when the seeds of both the cultivars of groundnut were inoculated with

A. flavus toxigenic strain, the penetration and establishment of the fungi in case of cv. J11 was slow

as compared to cv. JL 24. The results are in conformity with Ae gendy et al., (2001) who reported

cell wall fortifications such as deposition of callose, cellulose, lignin and structural proteins directly

below the point of attempted penetration to prevent the pathogen infection.

A thorough understanding of the host pathogen interaction between groundnut and A. flavus

may provide information that might be used to develop novel detection and screening methods. The

toxic properties of the aflatoxins produced by A. flavus are a major concern for growers and

consumers of groundnut. Elimination of the threat of infection due to A. flavus is important before

groundnut seeds goes into the storage. The present investigation reveals that A. flavus was seed

borne in nature and contaminated seeds were important source of inoculum for seed infection and

spread of the fungus from one seed to another during storage. SEM studies proved to be a reliable

method to detect the intercellular and intracellular hyphae of A. flavus which is undetectable with

the naked eye or by conventional light microscopy.

The present findings are in conformity with Achar et al. (2009) who reported that mycelium

of A. flavus was seen established in the host tissues both intercellularly and intracellularly and

continuous branching of young hyphae was seen in the groundnut seed. Rodriguez et al. (2007) who

reported the structure of aflatoxigenic molds and their identification up to species level and

Plate 4.5. Scanning electron micrographs of A. flavus growth in treated seeds of

groundnut resistant cv. J 11 at different days of incubation period

Day 9 Day 7

Day 3 Day 5

Plate 4.6. Scanning electron micrographs of A. flavus growth in treated seeds of

groundnut susceptible cultivar JL 24 at different days of incubation

period

Control Day 1

Day 9 Day 7

Day 3 Day 5

characterization. Hermetz et al. (2014) observed that establishment of seed borne nature of A. flavus

and its significance in seed and seedling infection using light microscopy, Scanning Electron

Microscopy and Transmission Electron Microscopy.

Scanning Electronic Microscopic (SEM) methodology enabled to observe the interaction of

fungi on surface of seeds and potential to increase the opportunities for teaching and learning in

Seed Pathology depending on the level of detail of observed structures. The adoption of this

technique in the future seed health analysis could be useful to identify fungal structures that enable

ensuring the implementation of correct diagnoses as well as to facilitate conduction of more detailed

taxonomic classification of seed-borne fungi.

4.5 EFFECT OF A. flavus INFECTION ON OIL CONTENT IN GROUNDNUT

RESISTANT cv. J 11

The effect of A. flavus infection on oil content in groundnut seeds were recorded. Oil

content was significantly differed in treated and untreated seed samples analyzed. The oil content

was gradually reduced at 1 to 56 days after incubation to an extent of 50.3 % to 41.3 % in the seeds

treated with A. flavus. In the untreated seeds (control) there was less reduction in oil content (50.6

% to 44.6 %) (Table 4.5).

4.5.1 Effect of A. flavus on oil content in groundnut susceptible cv. JL 24

The effect of A. flavus infection on oil content in groundnut seeds were assessed. Oil content

was significantly differed in treated and untreated seed samples analyzed. The oil content was

gradually reduced at 1 to 56 days after incubation. The reduction in oil content was high in treated

seeds (50.6 % to 32.3 %) as compared with untreated seeds (50.6 % to 36.9 %) (Fig 4.3).


compared to resistant groundnut cv. J 11 (9 %). While the reduction in oil content was low in the

untreated seeds of groundnut cv. JL 24 and groundnut cv. J 11 ( 13.7 % and 6 %).

The reduction in oil content might be attributed to lipids present in the seeds were primarily

neutral triglycerides and their hydrolysis to free fatty acids and glycerol were catalyzed by seed

borne fungi which caused the oxidation of fatty acids and inactivation of enzymes. This might be

one of the reasons for the reduction in oil content in the groundnut cultivars. The present results are

in agreement with Deshpande and Pancholy (1979), Bhattacharya and Raha (2002) and

Narayanswamy (2003) who reported that significant changes in oil content in the inoculated

groundnut seed samples with the advancement in the storage period.

Table 4.5. Effect of A. flavus infection on oil content (%) in treated and untreated seeds of

groundnut cvs. J 11 and JL 24

S. No Interval

(Days)

Oil content

(%)

cv. J 11 cv. JL 24

Treated Untreated Treated Untreated

1 1 50.3 50.6 50.6 50.6

2 3 47.2 48.4 47.9 47.9

3 7 47.1 47.5 46.8 47.8

4 14 47.0 47.5 46.8 47.5

5 21 45.8 47.4 46.4 47.3

6 28 45.8 47.4 46.3 47.0

7 35 45.5 47.2 43.5 42.5

8 42 45.5 46.1 38.3 39.3

9 49 44.8 45.8 34.9 38.0

10 56 41.3 44.6 32.3 36.9

SE (m) ± 1.29 0.91 0.73 0.93

CD at 5 % 3.80 2.70 2.17 2.76

Table 4.6. Effect of A. flavus infection on protein content (%) in treated and untreated seeds

of groundnut cvs. J 11 and JL 24

S. No Interval

(Days)

Protein content

(%)

cv. J 11 cv. JL 24


1 1 33.6 33.2 41.2 40.1

2 3 33.2 31.0 41.0 39.9

3 7 33.0 31.0 39.8 37.1

4 14 32.4 30.2 39.8 36.4

5 21 31.4 29.7 38.9 36.4

6 28 28.9 29.2 34.9 36.3

7 35 28.8 29.2 33.0 35.7

8 42 28.5 28.8 29.6 29.9

9 49 27.6 28.1 29.4 29.4

10 56 27.1 28.1 24.9 25.9

SE (m) ± 1.19 0.68 1.62 1.57

CD at 5 % 3.52 2.03 4.80 4.65

Fig 4.3. Oil content in treated and untreated seed samples of groundnut cultivars

0

10

20

30

40

50

60

50.3 47.2 47.1 47 45.8 45.8 45.5 45.5 44.8 41.3

Oil

con

ten

t (%

)

Interval

(Days)

cv. J 11 (Untreated)

cv. JL 24 (Treated)

cv. JL 24 (Untreated)


Fig 4.4. Protein content in treated and untreated seed samples of groundnut cultivars

0

5

10

15

20

25

30

35

40

45

1 3 7 14 21 28 35 42 49 56

Pro

tein

con

ten

t (%

)

Interval

(Days)

cv. J 11 (Treated)


cv. JL 24 (Treated)


4.6 EFFECT OF A. flavus ON PROTEIN CONTENT IN GROUNDNUT

RESISTANT cv. J 11

The effect of A. flavus infection on protein content in groundnut cv. J11 was recorded.

Protein content was significantly differed in treated and untreated seed samples analyzed. The

protein content was gradually reduced at 1 to 56 days after incubation. It was ranged from 33.6 %

to 27.1 % in the treated seeds and untreated seeds (33.2 % to 28.1 %) (Table 4.6).

4.6.1 Effect of A. flavus on protein content in groundnut susceptible cv. JL 24

The effect of A. flavus infection on protein content of groundnut cv. JL 24 was recorded.

Protein content was significantly differed in treated and untreated seed samples analyzed. The

protein content was gradually reduced from 1 to 56 days after incubation and ranging from 41.2 %

to 24.9 % in the seeds treated with A. flavus where-as in the untreated seeds (control) recorded less

reduction in protein content 40.1 % to 25.9 % (Table 4.6) (Fig 4.4).

Overall the per cent reduction in the protein content was found high in susceptible

groundnut cv. JL 24 (16.3 %) as compared to resistant groundnut cv. J 11 (6.5 %). While the

reduction in protein content was low in the untreated seeds of groundnut cvs. JL 24 and J 11 (14.2

% & 5.1%). The present results revealed that the rate of depletion in total protein was significantly

differed. It might be attributed that protein served as a primary source of readily available carbon

and nitrogen for growth and metabolism of the invading fungi. Loss in protein content during the

early phase of invasion and incubation indicated that proteolysis and formation of simpler

compounds such as amino acids which were utilized by the fungi. Similar trend of reduction in

protein content in the groundnut due to storage fungi was reported earlier by Narayanswamy

(2003), Adiver et al. (2015), Rammorthy and Karivarataraju (1989), Braccini et al. (2000),

Ushamalini et al. (1998), Kakde and Chavan (2011) and Bilgrami et al. (1976).

4.7 EFFECT OF A. flavus ON SATURATED FATTY ACIDS (PALMITIC

AND) IN GROUNDNUT RESISTANT cv. J 11 AND SUSCEPTIBLE cv.

JL 24

The effect of A. flavus infection on palmitic acid content of groundnut seeds were recorded. The

palmitic acid content in resistant cv. J 11 was gradually increased from 1 to 56 days after incubation. In the

treated seeds the increase in palmitic acid content was high (11 % to 14.9 %) as compared with untreated

seeds (control) (11 % to 13.9 %). Where as in susceptible cv. JL 24 the

Table 4.7. Effect of A. flavus infection on saturated fatty acids (Palmitic acid) content (%) in

treated and untreated seeds of groundnut cvs. J 11 and JL 24

S. No Interval

(Days)

Palmitic acid

(%)

cv. J 11 cv. JL 24


1 1 11.0 11.0 11.0 11.0

2 3 11.4 11.5 11.5 11.3

3 7 11.6 11.6 11.5 11.5

4 14 12.5 11.7 11.8 11.5

5 21 12.5 11.7 11.9 11.9

6 28 12.9 12.5 11.9 11.9

7 35 13.5 12.5 12.3 13.0

8 42 13.5 12.5 12.8 13.0

9 49 14.3 13.8 13.0 13.5

10 56 14.9 13.9 15.5 13.5

SE (m) ± 0.31 0.39 0.56 0.55

CD at 5 % 0.92 1.17 1.66 1.64

Table 4.8. Effect of A. flavus infection on saturated fatty acids (Stearic acid) content (%) in


S. No Interval

(Days)

Stearic acid

(%)

cv. J 11 cv. JL 24


1 1 0.72 0.74 0.72 0.71

2 3 0.79 0.83 0.96 0.72

3 7 1.36 0.95 0.95 1.36

4 14 1.67 0.96 1.19 1.67

5 21 1.91 1.68 2.48 1.91

6 28 2.20 2.17 2.84 2.30

7 35 2.37 2.30 3.35 2.31

8 42 2.40 2.48 3.45 2.40

9 49 2.63 2.56 4.89 2.63

10 56 3.65 2.68 5.31 2.71

SE (m) ± 0.20 0.30 0.39 0.19

CD at 5 % 0.61 0.90 1.16 0.57

Fig 4.5. Palmitic acid content in treated and untreated seed samples of groundnut cultivars

0

2

4

6

8

10

12

14

16

18

1 3 7 14 21 28 35 42 49 56

Palm

itic

aci

d (

%)

Interval

(Days)

cv. J 11 (Treated)


cv. JL 24 (Treated)


Fig 4.6. Stearic acid content in treated and untreated seed samples of groundnut cultivars

0

1

2

3

4

5

6

1 3 7 14 21 28 35 42 49 56

Ste

ari

c aci

d (

%)

Interval

(Days)

cv. J 11 (Treated)


cv. JL 24 (Treated)


increase in palmitic acid content of 11 % to 15.5 % in the treated seeds where as in the untreated

seeds there was slow increase of 11.0 % to 13.5 % (Table 4.7) (Fig 4.5).

4.7.1 Effect of A. flavus on saturated fatty acids (stearic acid) in groundnut resistant cv. J 11

and susceptible cv. JL 24

The effects of A. flavus on stearic acid content of groundnut seeds were recorded. The

increased levels of stearic acid content in resistant cv. J 11 was observed at 1 to 56 days after

incubation in treated seeds (0.72 % to 3.65 %) and untreated seeds (0.74 % to 2.68 %). Whereas, in

susceptible cv. JL 24 the stearic acid content was gradually increased from 1 to 56 days after

incubation. The per cent increase in stearic acid content was high (0.72 % to 5.31 %) in the treated

seeds as compared with the untreated seeds (0.71 % to 2.71%) (Table 4.8) (Fig 4.6).

Many of the fungi have been reported to cause physical and biochemical changes in crops

during storage as well as in releasing toxic substances which tend to limit their use and general

acceptability. In general, when the seeds were stored at higher moisture content the activity of

Aspergilli were found high which releases toxic metabolites into seeds. Presence of these toxic

substances in the seeds mainly affects seed quality and adversely making the seeds unfit for

consumption. An increased levels of free fatty acid contents in the groundnut cultivars over a period

of storage indicates the breakdown of triglycerides in groundnut oil leading to an eventual

deterioration of the seed quality and production of hydrolytic rancidity.

The present results are in conformity with Rammorthy and Karivarataraju (1989) who

reported a progressive increase in free fatty acid levels in the stored kernels than pods because there

was invasion of storage fungi. Jain (2008) also reported the increased levels of free fatty acid

content in the damaged seeds by fungal invasion. Mutegi et al. (2013) showed that rancidity was

significantly increased during storage. Storage fungi can change fat quality of groundnuts by

hydrolytic enzymes producing free fatty acids and glycerol. There was decrease in crude fat because

fungi might have degraded the lipids by lipase enzyme.

4.8 EFFECT OF A. flavus INFECTION ON UNSATURATED FATTY ACIDS

(LINOLEIC ACID) IN GROUNDNUT RESISTANT cv. J 11 AND

SUSCEPTIBLE cv. JL 24

The effect of A. flavus infection on linoleic acid content of groundnut seeds were recorded. The

linoleic acid content was gradually reduced at 1 to 56 days after incubation. The reduction in

linoleic acid was high in the treated seeds (42.1 % - 27.1 %) as compared with untreated seeds (43

Table 4.9. Effect of A. flavus infection on unsaturated fatty acids (Linoleic acid) content (%)

in treated and untreated seeds of groundnut cvs. J 11 and JL 24

S. No Interval

(Days)

Linoleic acid

(%)

cv. J 11 cv. JL 24


1 1 42.1 43.0 40.9 40.8

2 3 41.4 41.5 39.3 37.9

3 7 40.6 38.9 37.3 37.8

4 14 36.6 36.9 32.6 33.8

5 21 36.1 36.8 30.9 30.3

6 28 34.4 33.8 30.2 29.4

7 35 33.9 33.6 29.8 29.3

8 42 32.3 33.2 25.4 29.3

9 49 31.3 32.1 24.1 28.1

10 56 27.1 31.7 23.4 27.5

SE (m) ± 2.15 1.70 4.04 2.43

CD at 5 % 6.34 5.03 11.9 7.18

Table 4.10. Effect of A. flavus infection on unsaturated fatty acids (Oleic acid) content (%) in


S. No Interval

(Days)

Oleic acid

(%)

cv. J 11 cv. JL 24


1 1 43.9 43.9 41.7 42.0

2 3 42.8 42.9 37.8 41.1

3 7 40.3 41.3 36.9 40.6

4 14 40.2 41.2 33.6 38.0

5 21 40.1 40.6 30.3 36.9

6 28 40.1 40.2 29.4 36.8

7 35 39.7 40.1 29.0 36.8

8 42 38.9 39.7 27.7 35.9

9 49 38.6 38.7 27.5 34.6

10 56 29.9 37.9 25.1 33.8

SE (m) ± 1.70 1.02 1.98 1.82

CD at 5 % 5.01 3.03 5.85 5.38

Fig 4.7. Linoleic acid content in treated and untreated seed samples of groundnut cultivars

0

5

10

15

20

25

30

35

40

45

50

42.1 41.4 40.6 36.6 36.1 34.4 33.9 32.3 31.3 27.1

Lin

ole

ic a

cid

(%

)

Interval

(Days)


cv. JL 24 (Treated)



Fig 4.8. Oleic acid content in treated and untreated seed samples of groundnut cultivars

0

5

10

15

20

25

30

35

40

45

1 3 7 14 21 28 35 42 49 56

Ole

ic a

cid

(%

)

Interval

(Days)

cv. J 11 (Treated)


cv. JL 24 (Treated)


% to 31.7 %). Whereas, in susceptible cv. JL 24 the linoleic acid content was gradually reduced at 1

to 56 days after incubation. The rate of decrease was high in the treated seeds (40.9 % - 23.4 %) as

compared with untreated seeds (40.8 % - 27.5 %) (Table 4.9, Fig 4.7).

4.8.1 Effect of A. flavus on unsaturated fatty acids (oleic acid) in groundnut resistant cv. J 11

and susceptible cv. JL 24

The effect of A. flavus infection on oleic acid content in groundnut cv. J 11 was recorded. The

reduction in oleic acid content was observed in the treated seeds (43.9 % to 29.9 %) as compared

with untreated seeds (43.9 % to 37.9 %). Similar trend of reduction in oleic acid content were

observed in treated seeds of susceptible cv. JL 24 (41.7 % to 25.1 %) and untreated seeds (42.0 % to

33.8 %) (Table 4.10, Fig 4.8).

The reduction in linoleic and oleic acid contents were high in the treated seeds as compared to

the untreated seeds.

The per cent reduction in the unsaturated fatty acids like linoleic and oleic acids were high in

susceptible groundnut cv. JL 24 (17.5 % and 16.6 %) as compared to resistant groundnut cv. J 11

(15 % and 14 %). Whereas, in the untreated seeds, the per cent reduction in linoleic and oleic acids

were found low (11.3 % and 6 %) in groundnut cv. J 11 and 13.3 % and 8.2 % in groundnut cv. JL

24, respectively.

These results are in conformity with Braccini et al. (2000) who reported that reduction in

unsaturated fatty acids, protein and lipid content of soybean.

4.9 EFFECT OF A. flavus ON AFLATOXIN CONTET OF GROUNDNUT

RESISTANT cv. J 11 AND SUSCEPTIBLE cv. JL 24

The level of aflatoxin contents was increased at 1 to 56 days after incubation in the treated

seeds of groundnut cv. J 11 and cv. JL 24. Aflatoxin content of 2.15µg/kg to 2861.3 µg/kg in the

treated seeds and in the untreated seeds 2.15 µg/kg to 14.7 µg/kg were recorded in resistant cv. J 11.

The aflatoxin content was found high in susceptible cv. JL 24 (63.4 µg/kg to 4077.1 µg/kg) in the

treated seeds as compared to untreated seeds (2.15 µg/kg to 21.1 µg/kg) were recorded at different

days of incubation period.

In the present study, susceptible groundnut cv. JL 24 recorded increased aflatoxin levels (63.4

µg/kg to 4077.1 µg/kg) as compared to groundnut cv. J 11 (2.15 µg/kg to 2861.3 µg/kg) (Fig

Table 4.11. Effect of A. flavus infection on aflatoxin content (µg kg-1) in treated and untreated

seeds of groundnut cvs. J 11 and JL 24 at different days of incubation period.

S.

No

Interval

(Days)

cv. J 11 cv. JL 24


1 1 2.15

(1.62)

2.15

(1.76)

63.4

(6.03)

2.15

(1.76)

2 3 37.9

(6.10)

3.61

(2.14)

89.9

(9.40)

3.94

(2.22)

3 7 74.7

(8.60)

6.04

(2.64)

186.0

(13.2)

6.37

(2.70)

4 14 87.7

(8.43)

6.76

(2.75)

639.5

(20.4)

6.76

(2.75)

5 21 220.4

(12.0)

8.77

(2.98)

635.0

(23.7)

9.43

(3.06)

6 28 278.2

(16.5)

11.5

(3.44)

963.9

(29.9)

9.84

(3.23)

7 35 338.4

(17.5)

12.9

(3.72)

1111.7

(33.0)

12.9

(3.72)

8 42 850.9

(28.4)

13.8

(3.67)

1303.7

(34.4)

13.8

(3.67)

9 49 2182.6

(37.3)

14.0

(3.86)

2367.3

(48.5)

15.6

(4.07)

10 56 2861.3

(45.4)

14.7

(3.97)

4077.1

(63.8)

21.1

(4.66)

SE (m) ± 9.37 0.49 5.26 1.27

CD at 5 % 27.6 1.23 15.5 0.42

Figures in the parenthesis are square root transformed values. Each value is means of three

replications.

Fig 4.9. Effect of A. flavus infection on aflatoxin content in treated and untreated seeds of groundnut cvs. J 11 and JL 24 at different days of

incubation period

0

500

1000

1500

2000

2500

3000

3500

4000

4500

1 3 7 14 21 28 35 42 49 56

Afl

ato

xin

(µ

g k

g-1

)

Interval

(Days)

cv. J 11 (Treated)


cv. JL 24 (Treated)


4.9). Whereas, in the untreated seeds the detected aflatoxin content was within the permissible

levels in both resistant cv. J 11 (2.15 µg/kg to 14.7 µg/kg) and susceptible cv. JL 24 (2.15 µg/kg to

21.1 µg/kg).

The present results are in agreement with the findings of Yu et al. (2004), Hameeda et

al. (2006), Alemayehu et al. (2012) and Wartu et al. (2015) who reported that A. flavus was the

most predominant species responsible for aflatoxin contamination of crops prior to harvest or

during storage. The findings of Fabri et al. (1983), Passi et al. (1984), Doehlert et al. (1993) and

Burrow et al. (1997) indicated that fatty acid composition might directly or indirectly affects

aflatoxin biosynthesis.

4.10 EVALUATION OF SEED TREATMENTS WITH BIOAGENTS AND

FUNGICIDES AGAINST A. flavus IN GROUNDNUT SUSCEPTIBLE

cv.JL 24 UNDER GLASSHOUSE CONDITIONS

The results revealed that significant differences in different seed treatments were observed

as compared to untreated and pathogen treated seeds (Table 4.13). The germination percentage was

significantly improved in all the seed treatments as compared to untreated seeds. Among the seed

treatments, groundnut seeds treated with T. harzianum recorded higher seed germination (96 %)

followed by seed treatment with T. viride (91 %) and it was found on par with seeds treated with P.

fluorescens (88.2 %). The other seed treatments viz., carbendazim (81 %) and mancozeb (73.5 %)

were also found effective in increasing the seed germination over untreated seeds (65 %) and

pathogen treated seeds (54.5 %) (Fig 4.10) (Table 4.13).

Significant differences in plant height due to different seed treatments in groundnut seeds

were observed when compared with untreated and pathogen treated seeds. However, seed treatment

with T. harzianum recorded higher plant height (4.75, 12.9 and 14.1 cm) followed by seed treatment

with T. viride (4.10, 11.5 and 13.5 cm) and it was found on par with seeds treated with P.

fluorescens (3.40, 10.2 and 10.8) at 15, 30 and 45 DAS. The other seed treatments, carbendazim

(2.77, 8.02 and 9.21 cm) and mancozeb (2.72, 6.92 and 8.43 cm) were also found effective in

increasing plant height over untreated seeds (2.67, 6.62 and 7.37 cm) and pathogen treated seeds

(2.57, 5.65, 6.41 cm) at @ 15, 30 and 45 DAS (Fig 4.11) (Table 4.13).

Yield per plant was significantly improved in all the seed treatments as compared to treated and

untreated seeds. Among the seed treatments, groundnut seeds treated with T. harzianum recorded

higher pod yield per plant (4.6) followed by seed treatment with T. viride (4.2) and it was found on

par with seeds treated with P. fluorescens (4.1). Seeds treated with carbendazim (3.65)

Table 4.13 Evaluation of seed treatments with bioagents and fungicides against A. flavus under glasshouse conditions

S. No Treatments

Dosage

(g/kg)

Germination

(%)

Plant height

15 DAS

(cm)

Plant height

30 DAS

(cm)

Plant height

45 DAS

(cm)

Pod yield per

plant

(g)

1 Trichoderma harzianum 10 96.0

(79.7) 4.75 12.9 14.1 4.60

2 Trichoderma viride 10 91.0

(73.4) 4.10 11.5 13.5

4.20

3 Pseudomonas flourescens 10 88.2

(69.9) 3.40 10.2 10.8

4.10

4 Mancozeb 2.5 73.5

(59.0) 2.72 6.92 8.43 3.37

5 Carbendazim 2.0 81.0

(64.1) 2.77 8.02 9.21

3.65

6 Inoculated

control/Treated seeds

54.5

(47.5) 2.57 5.65 6.41

2.55

7 Uninoculated

control/untreated seeds

65.0

(53.7) 2.67 6.62 7.37 2.92

SEm ±

1.49 0.34 0.80 0.78 0.09

CD at 5 %

4.43 1.01 2.36 2.31 0.27

Figures in parenthesis are angular transformed values. Each value is mean of four replications.

.

Fig. 4.10. Evaluation of seed treatments with bioagents and fungicides against A. flavus on

germination

T1 - Trichoderma harzianum T6 - Inoculated control/Treated seeds

T2 - Trichoderma viride T7 - Uninoculated control/untreated seeds

T3 - Pseudomonas flourescens

T4 - Mancozeb

T5 - Carbendazim

0

20

40

60

80

100

120

T1 T2 T3 T4 T5 T6 T7

Ger

min

ati

on

(%

)

Seed treatments

Fig. 4.11. Evaluation of seed treatments with bioagents and fungicides against A. flavus on

plant height




T4 - Mancozeb

T5 - Carbendazim

0

2

4

6

8

10

12

14

16

T1 T2 T3 T4 T5 T6 T7

Pla

nt

hei

gh

t (c

m)

Seed treatments

15 Days

30 Days

45 Days

Fig. 4.12. Evaluation of seed treatments with bioagents and fungicides against A. flavus

on yield




T4 - Mancozeb

T5 - Carbendazim

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

T1 T2 T3 T4 T5 T6 T7

Pod

yie

ld p

lan

t-1

Seed treatmentss

Plate 4.7. Evaluation of seed treatments using bioagents and fungicides in groundnut

susceptible cv. JL 24 under glasshouse conditions

and mancozeb (3.37 ) were also found effective in increasing pod yield over untreated seeds (2.92)

and pathogen treated seeds (2.55) (Fig 4.12).

The results are in conformity with the earlier findings of Divakara et al. (2014) who reported

that seed treatment with talc based powder formulations of antagonist rhizobacteria and

Trichoderma sp. improved crop yield and reduced aflatoxin production in sorghum and ensuring

high economic returns. Dey et al. (2004) also observed that improvement in the growth, yield and

nutrient uptake in groundnut cultivar JL 24 in pots prior inoculated with PGPR isolates. Raju et al.

(1999) reported that formulations of P. fluorescens were effective in reducing F. moniliforme

infection and also increasing the germination, vigour index and field emergence.

4.10.1 Estimation of aflatoxin content in the harvested seeds of groundnut cv. JL 24

Groundnut seeds of cv. JL 24 treated with bioagents and fungicides were evaluated under

glasshouse conditions. The aflatoxin levels through ELISA method in the harvested produce of

different seed treatments were assessed. The presence of aflatoxin was detected in pathogen treated

seeds (1.38 µg/kg) and untreated seeds (0.69 µg/kg) which is below permissible levels (Table 4.12).

The present results are in conformity with Ajitkumar et al. (2011) who reported that T.

harzianum inhibited the growth of A. flavus to an extent of 70% followed by T. viride (68.9 %).The

beneficial effects of seed treatments with bioagents and fungicides in minimizing the pre harvest

infection in groundnut was reported earlier by Mixon et al. (1984) who stated that treatment of

groundnut pods with T. harzianum reduced the aflatoxin contamination. The present results were

also in agreement with Sanskrit et al. (2015) who reported that P. fluorescens showed inhibitory

activity against A. flavus. Use of biocontrol agents in aflatoxin management was also suggested by

Vijay Krishna Kumar et al. (2002). The present results are similar with Rathod et al, (2010) who

found that carbendazim was found effective against storage rot of groundnut caused by A. flavus.

Yitayih et al. (2013) also proved that seed treatments with carbendazim and mancozeb +

carbendazim were found effective in reducing the population densities of A. flavus. Alemayehu et

al. (2012) also reported that the total aflatoxin levels in Aspergillus flavus positive samples of

groundnut seed varied between 15 and 11865 µg/kg.

4.12. Estimation of Aflatoxin content (µg kg-1) in the harvested produce of groundnut cv. JL

24 treated with bioagents and fungicides

S. No Treatments

Dosage

(g kg-1)

Aflatoxin

content

(µg kg-1)

1 Trichoderma harzianum 10 0

(1.00)

2 Trichoderma viride 10 0

(1.00)

3 Pseudomonas flourescens 10 0

(1.00)

4 Mancozeb 2.5 0

(1.00)

5 Carbendazim 2 0

(1.00)

6 Inoculated control/Treated seeds 1.38

(1.29)

7 Uninoculated control/untreated seeds 0.69

(1.53)

SEm ± 0.12

CD at 5 % 0.04

Figures in the parenthesis are square root transformed values. Each value is means of four

replications.

CHAPTER V

SUMMARY AND CONCLUSIONS

Groundnut (Arachis hypogaea L.) is an important oil seed crop in India. It contains oil to an

extent of 48 - 51 %. The major problem associated with groundnut is aflatoxin contamination. It is

mainly caused by Aspergillus flavus and Aspergillus parasiticus. Keeping this in view, the present

findings pertaining to the investigations was carried out on detection and identification of seed

mycoflora, mode of entry of A. flavus into groundnut seed, effect of A. flavus on seed and oil quality

and to study the efficacy of bioagents and fungicides in the management of A. flavus in groundnut.

The results obtained from the present investigation are summarised as follows:

A total of seventy two groundnut (72) pod samples comprising farmer samples (36) and

market samples (36) were collected from major groundnut growing districts of Telangana state

during 2015 - 2016. The seed samples were analysed for seed health by agar plate method as per

ISTA (1996). Significant differences in occurrence of total number of fungal colonies due to

location and source of seed samples were observed. Irrespective of the districts, total per cent

occurrence of seed mycoflora was found high in farmer samples (92.4 %) over market samples

(45.3 %). Out of four districts, samples of Mahabubnagar district (47.1 % & 26.2 %) followed by

Warangal district (43.5 % & 24.6 %) recorded more total number of fungal colonies in farmer and

market samples. Irrespective of the samples, occurrence of six fungal flora viz., A. flavus, A. niger,

Fusarium sp. Alternaria sp. Macrophomina sp. Penicillium sp. Among them, A. flavus (43.2 %), A.

niger (26.7 %) were found predominant in both farmer and market samples.

External seed colonization due to A. flavus in groundnut resistant cv. J 11 and susceptible

cv. JL 24 were observed at different days of incubation period. Resistant cv. J 11 inoculated with A.

flavus colonized the seeds with severity score of 1, 2, 3, 4 and susceptible cv. JL 24 inoculated with

A. flavus colonized the seeds with a severity of 2, 3, 4, 4 at 3, 5, 7 and 9 days of incubation period.

The mode of entry of pathogen into groundnut seed was studied by Scanning Electron

Microscopy. Groundnut seeds of resistant and susceptible cultivars cvs. J 11 and JL 24 were

inoculated with A. flavus toxigenic strain penetration and establishment of the pathogen in case of

cv. J 11 was slow as compared to cv. JL 24. Irrespective of the cultivars, presence of pathogenic

mycelium on the damaged seed coat with fractured discontinuous epidermis and loose broken cell

junctions between epidermal cells were observed. In addition to this discontinuation, disorganized

parenchyma, broken and missing cell walls and total depletion of storage protein were observed.

Whereas, in untreated seeds of both the cultivars under SEM exhibited well organized, unruptured

cell walls with minimal intercellular space and abundant storage protein. The present results

revealed that A. flavus was seed borne in nature and contaminated seeds served as an important

source of seed infection and spread of the pathogen during storage.

Effect of A. flavus infection on oil content, protein content and unsaturated fatty acid profile

were reduced and increase in saturated fatty acid profile were observed over a period of incubation

in treated seeds and untreated seeds of both resistant and susceptible groundnut cultivars.


compared to resistant groundnut cv. J 11 (9 %). While the reduction in oil content was less in the

untreated seeds of groundnut cv. JL 24 and groundnut cv. J 11 ( 13.7% and 6%). Overall the per

cent reduction in the protein content was found high in susceptible groundnut cv. JL 24 (16.3 %) as

compared to resistant groundnut cv. J 11 (6.5 %). While the reduction in protein content was less in

the untreated seeds of groundnut cvs. JL 24 and J 11 (14.2 % & 5.1 %).

The increase levels of saturated fatty acids viz., palmitic and stearic acids were high in

susceptible cv. JL 24 (4.5 % & 4.5 %) as compared to resistant cv. J 11 (3.9 % & 2.93 %). Where as

in untreated seeds, the increased levels in palmitic and stearic acids were found low (2.5 & 2 %) in

cv. J 11 and 2.9 % and 1.94 % in groundnut cv. JL 24 respectively.

The per cent reduction in the unsaturated fatty acids linoleic and oleic acids were high in

susceptible groundnut cv. JL 24 (17.5 % and 16.6 %) as compared to resistant groundnut cv. J 11

(15 % and 14 %). Whereas, in the untreated seeds, the per cent reduction in linoleic and oleic acids

were found low (11.3 % and 6 %) in groundnut cv. J 11 and 13.3 % and 8.2 % in groundnut cv. JL

24, respectively.

The aflatoxin content at 1 to 56 days after incubation were increased from 2.15 µg/kg -

2861.3 µg/kg & 63.4 µg/kg - 4077.1 µg/kg in groundnut cv. J 11 and JL 24 respectively. When it

was compared with in untreated there was low levels of aflatoxin 2.15 µg/kg – 14.7 µg/kg in cv. J

11 and 2.15 µg/kg - 21.1 µg/kg in cv. JL 24 were recorded.

The efficacy of seed treatments against seed borne A. flavus were evaluated under glasshouse

conditions. Groundnut seeds treated with T. harzianum was significantly superior in recording

higher seed germination (96 %), plant height (4.75, 12.9 and 14.1 cm) and yield (4.60 g) followed

by T. viride (91 %, 4.10, 11.5 and 13.5 cm, 4.20 g) which was on par with P. fluorescens (88.2 %,

3.40, 10.2 and 10.8 cm 4.10 g). The remaining seed treatments were also found effective in

improving seed germination, plant height and yield in seeds treated with carbendazim (81 %, 2.77,

8.02 and 9.21 cm, 3.65 g), mancozeb (73.5 %, 2.72, 6.92 and 8.43 cm, 3.37 g) over untreated (65 %,

2.67, 6.62 and 7.37 cm, 29.2) and pathogen treated seeds (54.5 %, 2.57, 5.65, 6.41 cm, 2.55 g) at

15, 30, 45 DAS. Aflatoxins were detected in pathogen treated seeds (1.38 µg/kg) and untreated

seeds which is better permissible level. While aflatoxin was not observed in the seed treated with

pathogen as T. harzianum, T. viride and P. fluorescens.

The following conclusions have been drawn from the investigations are as follows.

Groundnut farmer seed samples recorded high incidence of seed mycoflora over market

samples.

Out of four districts of seed samples collected, Mahabubnagar district samples recorded

maximum occurrence of more number of total fungal colonies.

Among the seed mycoflora, A. flavus was predominant fungi in farmers and market samples.

The surface colonization of A. flavus was more in susceptible cv. JL 24 as compared to

resistant groundnut cv. J 11.

Groundnut seeds inoculated with A. flavus toxigenic strain through SEM showed penetration

and establishment of the pathogen in case of J 11 is slow as compared to JL 24.

Effect of A. flavus on oil content, protein content and unsaturated fatty acid profile were

reduced and increased levels of saturated fatty acid profile over incubation period was

observed in treated and untreated seeds.

Groundnut seeds treated with T. harzianum was found effective in improving seed

germination, plant height and yield and also reducing the A. flavus infection.

LITERATURE CITED

Achar, P.N., Hermetz, K., Rao, S., Apkarian, R and Taylor, J. 2009. Microscopic studies on the

Aspergillus flavus infected kernels of commercial peanuts in Georgia. Ecotoxicology and

Environmental Safety. 72: 2115-2220.

Adiver, S.S and Kirankumar, M.G. 2015. Incidence of Aspergillus flavus L. Ex. Fries in groundnut

(Arachis hypogaea L.) samples and its impact on nutritive value of kernels. Karnataka

Journal of Agricultural Sciences. 28 (2): 224 - 227.

Afzal, H., Shazad, S and Nisa, S.Q.U. 2013. Morphological identification of Aspergillus species

from the soil of larkana district (SINDH, PAKISTAN). Asian Journal of Agricultural

Biology. 1 (3): 105-117.

Agag, B.I. March 2004. Mycotoxins in foods and feeds aflatoxins. Assiut University Bulletin

Environmental Resource. 7 (1): 173 - 206.

Ajitkumar, K., Naik, M.K and Savita, A.S. 2011. Reduction of aflatoxin contamination in chilli

using eco-friendly management practices. Journal of Plant Disease Sciences. 6 (2): 153 -

156.

Aldao, M.A.J., Carpinella, M., Corelli, M and Herrero, G.G. 1995. Competitive ELISA for

quantifying small amounts of aflatoxin B1. Food and Agricultural Immunology. 7: 307-314.

Alemayehu, C., Abdi, M., Amare, A. and Helge, S. 2012. Natural occurrence of aflatoxins in

groundnut (Arachis hypogaea L.) from eastern Ethiopia. Food Control. 30: 602-605.

Alves, M.C and Pozza, E.A. 2012. Scanning electron microscopy detection of seed-borne fungi in

blotter test. FORMATEX. 230-238.

Ameer, M., Venudevan, B and Jayanthi, M. 2013. Storage fungi in groundnut and the associate seed

quality deterioration - A review. Plant Pathology Journal. 12 (3): 127-134.

Anjaiah, V and Thakur, R.P. 2000. Biological control of Aflatoxin contamination in groundnut:

Potential antagonists and their characterization.

Anjaiah, V., Thakur, R.P and Koedam, N. 2006. Evaluation of bacteria and Trichoderma for

biocontrol of pre-harvest seed infection by Aspergillus flavus in groundnut. Biocontrol

Science and Technology. 16 (4): 431-436.

Anjaiah,V., Thakur, R.P and Rao, V.P. 2001. Molecular diversity in Trichoderma isolates with

potential for biocontrol of Aspergillus flavus infection in groundnut. International Arachis

News letter. 21: 31-33.

Asis, R., Di paola, R.D and Aldao, M.A.J. 2002. Determination of Aflatoxin B1 in Highly

Contaminated Peanut Samples Using HPLC and ELISA. Food and Agricultural

Immunology. 14: 201-208.

Assefa, D., Tear, M and Skinnes, H. 2012. Natural Occurrence of Toxigenic Fungi Species and

Aflatoxin in Freshly Harvested Groundnut Kernels in Tigray, Northern Ethiopia. Journal of

the drylands. 5 (1): 377-384.

Ayejuyo, O.O., Olowu, R. A., Agbaje, T.O., Atamenwan, M and Osundiya, M.O. 2011. Enzyme -

Linked Immunosorbent Assay of Aflatoxin B1 in Groundnut and Cereal Grains in Lagos,

Nigeria. Research Journal of Chemical Sciences. 1 (8): 1-5.

Bagwan, N.B. 2011. Eco-friendly management of aflatoxin B1 at pre-harvest level in groundnut

(Arachis hypogaea L.). International Journal of Plant Protection. 4 (1): 1-6.

Baig, M., Fatima, S., Kadam, V.B and Shaikh, Y. 2012. Utilization of antagonist against seed borne

fungi. Trends in Life Sciences. 1 (1): 42-46.

Bakhiet, S.E.A and Musa, A.A.A. 2011. Survey and determination of aflatoxin levels in stored

peanut in Sudan. Jordan Journal of Biological Sciences. 4 (1):13-20.

Bansod, B., Thakur, R. and Holser, R. 2015. Analysis of Peanut seed oil by NIR. American Journal

of Analytical Chemistry. 6: 917-922.

Batten G.D. 1998. Plant analysis using near infrared reflectance spectroscopy: the potential and the

limitations. Austrlian Journal of Experimental Agriculture. 38: 697-706

Begum, M.A.J., Venudevan, B and Jayanthi, M. 2013.Storage fungi in groundnut and associative

seed quality- A review. Plant pathology Journal.12 (3): 127-134.

Bennett, J. W. 2010. An overview of the genus Aspergillus. Molecular Biology and Genomics. 1-

17.

Bhattacharya, K and Raha, S. 2002. Deteriorative changes of maize, groundnut and soybean seeds

by fungi storage. Mycopathologia. 155: 135-141.

Bhushan, G., Sharma, S., Kumar, S and Sagar, P. 2013. Effect of Moisture Content on Seed

Microflora, Nutritive Value Deteriorate and Aflatoxin Contamination of Sorghum Vulgare

Seeds During Storage. Asian Journal of Biochemical and Pharmaceutical Research. 1 (4):

202-206.

Bilgrami, K.S., Prasad, T., Jamaluddin and Roy, A.K. 1976. Studies on the deterioration of some

pulses by fungi. Indian Phytopathology. 29: 574-577.

Boutrif, E and Canet, C. 1998. Mycotoxin prevention and control: FAO programmes. Revue de

Médecine Vétérinaire. 149: 681-694.

Braccini, A., Reis, M.S., Moreria, M.A., Sediyama, C.S and Scapim, C.A. 2000. Biochemical

changes associated to soybean seeds osmo conditioning during storage. Pesquisa

Agropecuária Brasileira. 35: 433-447.

Burrow, G.B., Nesbitt, T.C., Dunlap, J and Keller, N.P. 1997. Seed lipoxygenase products modulate

Aspergillus mycotoxin biosynthesis. Molecular Plant Microbe Interactions Journal. 10:

380- 387.

Chala, A., Mohammed, A., Ayalew, A and Skinnes, H. 2013. Natural occurrence of aflatoxins in

groundnut (Arachis hypogaea L.) from Eastern Ethiopia. Food Control. 30: 602-605.

Chavan, A.M. 2011. Nutritional changes in oil seeds due to Aspergillus spp. Journal of

Experimental Sciences. 2 (4): 29-31.

Chen, Y.C., Liao, C.D., Lin, H.Y., Chiueh, L.C and Shih, D.Y.C. 2013. Survey of aflatoxin

contamination in peanut products in Taiwan from 1997 to 2011. Journal of food and drug

analysis. 21: 247-252.

Coulibaly, O., Hell, K., Bandyopadhyay, R., Hounkponou, S and Leslie, J.F. 2008. Mycotoxins:

Detection Methods, Management, Public Health and Agricultural Trade. CAB International

Publishers, Wallingford.

Desai, S., Thakur, R.P., Rao, V.P and Anjaiah, V. 2000. Characterisation of isolates of Trichoderma

for biocontrol potential against Aspergillus flavus infection in groundnut. International

Arachis Newsletter. 20: 57-59.

Deshpande, A.S and Pancholy, S.K. 1979. Colonization and Biochemical changes in peanut seeds

infected with Aspergillus flavus. Peanut science. 6: 102-105.

http://www.scielo.br/pab

http://www.scielo.br/pab

Dey, R., Pal, K.K., Bhatt, D.M and Chauhan, S.M. 2004. Growth promotion and yield enhancement

ofpeanut (Arachis hypogaea L.) by application of plant growth-promoting rhizobacteria.

Microbiological Research. 159: 371-394.

Dharmaputra, O.S. 2003. Control of aflatoxigenic Aspergillus flavus in peanuts using

nonaflatoxigenic A. flavus, A. niger and Trichoderma harzianum. Biotropia. 21: 32-44.

Ding, X., Li, P., Bai, Y and Zhou, H. 2012. Aflatoxin B1in post-harvest peanuts and dietary risk in

China. Food Control. 23: 143-148.

Directorate of Economics and Statistics, Government of Telangana, Statistical year book of

Telangana, 2015.

Divakara, S.T., Aiyaz, M., Hariprasad, M., Nayaka, S.C and Niranjana, S.R. 2014. Aspergillus

flavus infection and aflatoxincontamination in sorghum seeds and their biological

management. Archives of Phytopathology and Plant Protection. 47 (17): 2141-2156.

Doehlert, D.C., Wicklow, D.T and Gardner, H.W. 1993. Evidence implicating the lipoxygenase

pathway in providing resistance to soybeans against Aspergillus flavus. Phytopatholy. 83:

1473-1477.

El‚AeGendy, W., 2001. Rapid deposition of wheat cell wall structural proteins in response to

Fusarium‚Aederived elicitors. Journal of Experimental Botany. 52 (354): 85-90.

Eva G. Lizárraga-Paulín, Ernesto Moreno-Martínez and Susana P. Miranda-Castro. 2007.

Aflatoxins and Their Impact on Human and Animal Health: An Emerging Problem.

Biochemistry and Molecular Biology.

Fabri, A.A., Fanelli, C., Fanfili, G., Passi, S and Fasella, P. 1983. Lipoperoxidation and aflatoxin

biosynthesis by Aspergillus parasiticus and A. flavus. Journal of General Microbiology.

129: 3447-3452.

Fagbohun, E.D and Faleye, O.S. 2012. The nutritional and mycoflora changes during storage of

groundnut (Arachis hypogeal L). International Journal of Agronomy and Agricultural

Research. 2 (6): 15-22.

FAO (Food and Agriculture Organization) Statistical Database. 2013.

(http://faostat.fao.org/faostat/).

http://faostat.fao.org/faostat/

Gachomo, E.W and Kotchoni. 2008. The use of Trichoderma harzianum and T. viride as potential

biocontrol agent against peanut mycoflora and their effectiveness reducing aflatoxin in

reducing aflatoxin contamination of infected kernels. Biotechnology. 7 (3): 439-447.

Ginting, E and Rahmianna, A. 2015. Infection of Aspergillus flavus and Physical Quality of Peanuts

Collected from Farmers, Local Markets, and Processors. Procedia Food Science. 3: 280-

288.

Giray, B., Atasayar, S and Sahin, G. 2009. Determination of ochratoxin A and total aflatoxin levels

in corn samples from Turkey by enzyme-linked immunosorbent assay. Mycotoxins

Research. 25: 113-116.

Goldblatt, L.A. 1969. Aflatoxin. Scientific background, control and implications. Academic press,

New York. 472.

Gomez, K.A and Gomez, A.A. 1984. Statistical procedures for agricultural research (second

edition) John Wiley and sons, New York.

Guchi, E 2015. Aflatoxin Contamination in Groundnut (Arachishypogaea L.) Caused by

Aspergillus Species in Ethiopia. Journal of Applied & Environmental Microbiology. 3 (1):

11-19.

Guchi, E., Ayalew, A., Dejene, M., Ketema, M., Asalf, B and Fininsa, C. 2014. Occurrence of

Aspergillus species in groundnut (Arachis hypogaea L.) along the value chain in different

agro ecological zones of Eastern Ethiopia. Journal of Applied and Environmental

Microbiology. 2 (6): 309-317.

Hameeda, B., Rupela, O.P and Reddy. 2006. Antagonistic activity of bacteria inhibiting against soil

borne plant pathogenic fungi. Indian Journal of Microbiology. 46 (4): 289-396.

Hermetz, K., Achar, P., Apkarian, R.P., Taylor, J. 2014. Scanning and Transmission Electrin

Microscopy of Aspergillus flavus in Georgia peanuts.

Holbrook, C.C., Wilson, D.M., Matheron, M.E., Hunter, J.E., Knauft, D.A and Gorbet, D.W. 2000.

Aspergillus colonization and aflatoxin contamination in peanut genotypes with reduced

linoleic acid composition. Plant Disease. 84 (2): 148-150.

Hong, L.S., Yusof, N.I.M and Ling, H.M. 2010. Determination of Aflatoxins B1 and B2 in Peanuts

and Corn Based Products. Sains Malaysiana. 3(5): 731-735.

Ibiam, O. F. A and Egwu, B.N. 2011. Post-harvest seed-borne diseases associated with the seeds of

three varieties of groundnuts, (Arachis hypogaea L.) Nwakara, Kaki, and Campalla.

Agriculture and Biology Journal of North America. 2 (4): 598-602.

Iheanacho, H.E., Njobeh, P.B., Dutton, F.M., Steenkamp, P.A., Steenkamp, L., Mthombeni, J.Q.,

Daru, B.H and Makun, A.H. 2014. Morphological and molecular identification of

filamentous Aspergillus flavus and Aspergillus parasiticus isolated from compound feeds in

South Africa. Food Microbiology. 44: 180-184.

ISTA. 1996. International Rules of Seed Testing. Proceedings of International Seed Testing

Association. 32: 565-589.

Jain, P.C., 2008. Applied microbiology: Microbial degradation of grains, oil seeds, textiles, wood,

corrosion of metals and bioleaching of mineral ores. Department of Applied Microbiology &

Biotechnology. 1-37.

Kakde, R.B and Chavan, A.M. 2011. Deteriorative changes in oilseeds due to storage fungi and

efficacy of botanicals. Current Botany. 2 (1): 17-22.

Kalyani, S., Kiran, S., Surekha, M and Reddy, S.M. 2014. Incidence of mycotoxigenic fungi on

peanut seeds of Warangal district of A.P. Indian Journal of Applied Research. 4 (2): 4-6.

Khorasgani, Z.N., Nakisa, A and Farshpira, N.R. 2013. The Occurrence of Aflatoxins in Peanuts in

Supermarkets in Ahvaz, Iran. 2013. Journal of Food Research. 2 (1): 94-100.

Kishore, G.K., Pande, S., Manjula, K.J., Rao, N and Thomas, D. 2002. Occurrence of mycotoxins

and toxigenic fungi in Groundnut (Arachis hypogaea L.) seeds in Andhra Pradesh, India.

Plant Pathology Journal. 18 (4): 204-209.

Kolosova, A.Y., Shim, W., Yang, Z., Eremin, S.A and Chung, D. 2006. Direct competitive ELISA

based on a monoclonal antibody for detection of aflatoxin B1. Stabilization of ELISA kit

components and application to grain samples. Analytical and Bioanalytical Chemistry. 384:

286-294.

Li, P.Q., Zhang, W., Zhang, J., Zhang, X., Chen, J., Jiang. 2009. Development of class-specific

monoclonal antibody-based ELISA for aflatoxins in peanut. Food Chemistry. 115: 313-317.

Lia Qi fang. 2006. Study on detection of Aflatoxin B1 by ELISA and TLC.

Liu, Y and Wu, F. 2010. Global Burden of Aflatoxin-Induced Hepatocellular Carcinoma: A Risk

Assessment. Environmental Health Perspectives.118 (6): 818-824.

Lubulwa, G and Davis, J. 1994. Estimating the social costs of the impacts of fungi and aflatoxins in

maize and peanuts. In: Highley, E., Wright, E.J., Banks, H.J. & Champ, B.R. (Eds)

Proceedings of the 6th International Conference in Stored-Product Protection. 1017-1042.

Lumpungu, K., Baelenge, B and Bitijula, M. 1989. The effect of groundnut seed coat on the

development of pathogenic fungi. Tropicitura. 7 (4): 128-131.

Mahrous, S.R. 2007. Chemical Properties of Aspergillus flavus-Infected Soybean Seeds Exposed to

γ-Irradiation during Storage. International Journal of Agricultural Biology. 9 (2): 231-238.

Mathur, P.B., Sunkara, S., Bhatnagar Panwar, M., Waliyar, F and Sharma, K.K. 2015.

Biotechnological advances for combating Aspergillus flavus and aflatoxin contamination

in crops. Plant Science.

Mehan, V.K., McDonald, D and Rajagopalan, K. 1987. Resistance of Peanut Genotypes to Seed

Infection by Aspergillus flavus in Field Trials in India. Peanut Science. 14: 17-21.

Mixon, A.C and Rogers, K.M. 1973. Peanut accessions resistant to seed infection by Aspergillus

flavus. Agronomy Journal. 65: 560-562.

Mixon, A.C., Bell, D.K and Wilson, D.M. 1984. Effect of Chemical and Biological agents on the

incidence of Aspergillus and aflatoxin contamination of peanut seed. Phytopathology. 74:

1440-1444.

Mohamed, A.K., Almoammar, H and Kamle A. Abd-elsalam. 2015. Mycological, Biochemical, and

Molecular methods for detection of Aflatoxgenic Aspergilli from Peanut kernels. The

Philippine Agriculture Scientist. 98 (1): 52-59.

Mohammed, A and Chala, A. 2014. Incidence of Aspergillus contamination of groundnut (Arachis

hypogaea L.) in Eastern Ethiopia. African Journal of Microbiology Research. 8 (8): 759-

765.

Mohan, G., Bentur, K. G., Parameshwarappa and Appa Rao, 2003. In vitro screening of

confectionary groundnut genotypes for seed colonization by A. flavus, ISOR National

Seminar: Stress Management in oilseeds. 328-329.

Moreno, O. J., Kang. M. S. 1999. Aflatoxins in maize: the problem and genetic solutions. Plant

Breeding. 118: 1-16.

Mukherjee, P.S., Nandi, S.K and Nandi, B. 1992. Deteriorative changes in groundnut seeds in

storage. Journal of Mycopathological Research. 30 (2): 113-119.

Mutegi, C.K., Wagacha, J.M., Christie, M.E., Kimani, J and Karana L. 2013. Effect of storage

conditions on quality and aflatoxin contamination of peanuts (Arachis hypogaea L.).

International Journal of Agri Science. 3 (10): 746-758.

Nagpurne, V. S. N and Patwari, J. M. 2014. Seed borne mycoflora of groundnut. Trends in Life

Sciences. 3 (1): 4731-4735.

Nakai, V.K., Rocha, L.D.O., Gonacalez, E., Fonseca, H., Ortega, E.M.M and Corre, B. 2008.

Distribution of fungi and aflatoxins in a stored peanut variety. Food Chemistry. 106: 285-

290.

Naqvi, S.D.Y., Shiden, T., Merhawi, W and Mehret S. 2013. Identification of seed borne fungi on

farmer saved sorghum (Sorghum bicolor L.), pearl millet (Pennisetum glaucum L.) and

groundnut (Arachis hypogaea L.) seeds. Agricultural Science Research Journals. 3 (4): 107-

114.

Narayanaswamy, S. 2003. Effect of packaging and location of storage on groundnut seed.

Proceedings of the national Workshop on Groundnut seed Technology. 164-167.

Otsuki, T., Wilson, J.S and Sewadeh, M. 2001. Saving two in a billion: quantifying the trade effect

of European food safety standards on African exports. Food Policy. 26: 495-514.

Palumbo, J.D., O'Keeffe, T.L., Kattan, A., Abbas, H.K and Johnson, B.J. 2010. Inhibition

of Aspergillus flavus in soil by antagonistic Pseudomonas strains reduces the potential for

airborne spore dispersal. Biological Control. 100 (6): 532-538.

Passi, S., Nazzaro-porro, M., Fanelli, C., Fabri, A.A and Fasella, P. 1984. Role of lipoperoxidation

in aflatoxin production. Applied Microbiology and Biotechnology. 19: 186-190.

Patil, A.G., Oak, M.D., Taware, S.P., Tamhankar, S.A and Rao, V.S. 2010. Non destructive

estimation of fatty acid composition in soybean [Glycine max (L.) Merrill] seeds using Near-

Infrared Transmittance Spectroscopy. Food Chemistry. 120: 1210-1217.

Pitt, J.I and Hocking, A.D. 1997. Fungi and Food spoilage. London, Champan & Hall. 593.

Pushpalatha, K.C. 2013. Biocontrol Efficiency of Trichococcus spp., against Seed-borne Fungal

Pathogen of Peanut. International Journal of Biotechnology and Bioengineering Research. 4

(7): 665-670.

Radoi, A., Targa, M., Prieto-Simon, B and Marty, J.L. 2008. Enzyme-linked immunosorbent assay

(ELISA) based on superparamagnetic nanoparticles for aflatoxin M1 detection. Talanta. 10-

19.

Rahmianna, A.A and Yusnawan, E. 2015. Monitoring of aflatoxin contamination at market food

chain in east Java. Journal of Experimental Biology and Agricultural Sciences. 3 (4): 346-

352.

Rahmianna, A.A., Purnomo, J and Yusnawan, E. 2015. Assessment of groundnut varietal tolerant to

aflatoxin contamination in Indonesia. Procedia Food Science. 3: 330-339.

Rajarajan, P.N., Rajasekaran, K.M and Asha Devi, N.K. 2013. Isolation and quantification of

aflatoxin from Aspergillus flavus infected stored peanuts. Indian Journal of

Pharmaceutical and Biological Research. 1 (4): 76-80.

Raju, N.S., Niranjana, S.R., Janardhana, G.R., Prakash, H.S., Shetty, H.S. and Mathur, S.B. 1999.

Improvement of seed quality and field emergence of Fusarium moniliforme infected

sorghum seeds using biological agents. Journal of the science of Food and Agriculture. 79:

206-212.

Ramakrishna, N and Mehan, V.K. 1993. Direct and indirect competitive monoclonal antibody-

based ELISA of aflatoxin B1 in groundnut. Mycotoxin research. 9 (1): 53-63.

Ramamoorthy, K and Karivaratharaju, T.V. 1989. Influence of pod and kernel treatments on the

field performance of groundnut (Arachis hypogaea. L.) cv. Pol.2 after twelve months of

storage. Seeds Farms.15: 15-19.

Ramannujpatel, Patel, D.R and Pandey, A.K. 2014. Study on seed borne mycoflora of soybean,

sorghum and groundnut of different zones of Madhya Pradesh. International Journal of

Plant Protection. 7 (1): 9-14.

Rasheed, S., Darvar, S and Ghaffar, A. 2014. Location of fungi in groundnut seed. Pakistan Journal

of Botany. 36: 663-668.

Rasheed, S., Darvar, S., Ghaffar, A and Shakut, S.S. 2004. Seed borne mycoflora of groundnut.

Pakistan Journal of Botany. 36 (1): 199-202.

Rathod, L.R., Jadhav, M. D., Mane, S. K., Muley, S.M and Deshmukh, P. S. 2012. Seed borne

mycoflora of legume seeds. International Journal of Advanced Biotechnology and Research.

3 (1): 530-532.

Rathod, L.R., Jadhav, M.D., kanse, D.S., Patil, D.P., Gulhane, S.D and Deshmukh, P.S. 2010.

Effects of fungicides on seed borne pathogen of groundnut. International Journal of

Advanced Biotechnology and Research. 1 (1): 17-20.

Reddy, K.R.N., Reddy, C.S and Muralidharan, K. 2009. Potential of botanicals and biocontrol

agents on growth and aflatoxin production by Aspergillus flavus infecting rice grains.

Food Control. 20: 173-178.

Reddy, S.V., KiranMayi, D., Uma Reddy, M., Thirumala Devi, K and Reddy, D.V.R., 2001.

Aflatoxin B1 in different grades of chillies (Capsicum annum) as determined by indirect

competitive-ELISA. Food Additives and Contaminants. 18: 553-558.

Rodrigues, P., Soares, C., Kozakiewicz, Z., Paterson, R.R.M., Lima, N and Venâncio, A. 2007.

Identification and characterization of Aspergillus flavus and aflatoxins. Communicating

Current Research and Educational Topics and Trends in Applied Microbiology. 527-534.

Romero, I.J.L., Moreno, M.C., Martinez, A.F and Cerrato, R.F. 2000. Possibility of biological

control of Aspergillus flavus with Pseudomonas fluorescens on maize ear. Revista

Mexicana de Fitopatología. 18 (1): 50-54.

Sanskriti, R., Vijayalakshmi., Neelam, R., Ezilrani, P and Christopher, J.G. 2015. Biocontrol of

Aspergillus flavus in groundnut. Life Science Archives. 1 (1): 28-34.

Shephard G.S. 2003. Aflatoxin and food safety: recent African perspectives. Journal of Toxicology.

22 (2&3): 267-286.

Shephard, G.S. 2008. Impact of mycotoxins on human health in developing countries. 2008. Food

Additives and Contaminants. 25 (2): 146-151.

Strosnider, H., Azziz-Baumgartner, E., Banziger, M., Bhat, R.V., Breiman, R., Brune, M-N.,

DeCock, K., Groopman, J., Hell, K and Henry, S.H. 2006. Working group report: Public

health strategies for reducing aflatoxin exposure in developing countries. Environmental

Health Perspectives. 114: 1898-1903.

Sudha, S., Naik, M.K and Ajithkumar, K. 2013. An integrated approach for the reduction of

aflatoxin contamination in chilli (Capsicum annuum L.). Journal of Food Science and

Technology. 50 (1): 159-164.

http://www.ncbi.nlm.nih.gov/pubmed/?term=Shephard%20GS%5BAuthor%5D&cauthor=true&cauthor_uid=18286404

Sundaram, J., Kandala, C.V., Govindarajan, K.N and Subbiah, J. 2012. Sensing of Moisture

Content of In-Shell Peanuts by NIR Reflectance Spectroscopy. Journal of Sensor

Technology. 2: 1-7.

Sundaram, J., Kandala, C.V., Holser, R.A., Butts, C.L and Windham, W.R. 2010. Determination of

In-Shell Peanut Oil and Fatty Acid Composition Using Near-Infrared Reflectance

Spectroscopy. Journal of the American Oil Chemists' Society. 87: 1103- 1114.

Sylos, C. M. D. E., Rodriguez Amaya, D., Pinto, C., and Desylos, C. M. 1996. Comparison of

immunoassay and minicolumn chromatography for the screening of aflatoxins in groundnut

and maize. Alimentos-e-Nutricao. 7: 7-14.

Thakur, R.P., Rao, V.P and Subramanyam, K. 2003. Influence of biocontrol agents on population

density of Aspergillus flavus and kernel infection in groundnut. Indian Phytopathology.

56 (4): 408-412.

Thakur, R.P., Rao, V.P., Reddy, S.V., and Ferguson, M. 2000. Evaluation of wild Arachis

Germplasm Accessions for In Vitro Seed Colonization and Aflatoxin Production by

Aspergillus flavus. 20: 44-46.

Ushamalini, C., Rajappan, K and Gadharan, K. 1998. Changes in the constituents of cowpea due to

seed borne fungi. Indian Phytopathology. 51: 258-260.

Vasundara, P., Rangaswamy, V and Johnson, M. 2015. Effect of Seed Treating Pesticides with

Trichoderma viridae on Rhizosphere Mycoflora and Plant Biometrics at 75 DAS of

groundnut (Arachis hypogaea L.). International Journal of Current Microbiology and

Applied Science. 4 (4): 206-215

Verma, J.P., Yadav, J., Tiwari, K.N., Singh, V., 2010. Impact of plant growth promoting

rhizobacteria on crop production. International Journal of Agriculture Research. 5: 954-

983.

Vijay Krishna Kumar, K., Desai, S., Rao, V.P., Nur, H.A and Thakur, R.P. 2002. Evaluation of an

integrated aflatoxin contamination package to reduce pre-harvest seed infection by

Aspergillus flavus in groundnut. International Arachis Newsletter. 22: 42-44.

Waliyar, F., Ba, A., Isra, B.P., Hassan, H., Bonkoungou, S and Bosc, J.P. 1994. Sources of

resistance to Aspergillus flavus and aflatoxin contamination in groundnut genotypes in West

Africa. Plant Disease. 78 (7): 704-704.

http://link.springer.com/journal/11746

Waliyar, F., Kumar, P.L., Ntare, B.R., Diarra, B and Kodio, O. 2008. Pre-and post-harvest

management of aflatoxin contamination in peanuts. In Leslie J. F., Bandyopadhyay. R.,

Visconti. A. (eds.). Mycotoxins: Detection methods, management, public health and

agricultural trade. CABI. Wallingford. 209-218.

Waliyar, F., Umeh, V.C., Traore, A., Osiru, M., Ntare, B.R., Diarra, B., Kodio, O., Vijay Krishna

Kumar, K and Sudini, H. 2015. Prevalence and distribution of aflatoxin contamination in

groundnut (Arachis hypogaea L.) in Mali, West Africa. Crop Protection. 70: 1-7.

Wartu, J.R., Whong, C.M.Z., Umoh, V.J and Diya, A.W. 2015. Occurrence of Aflatoxin Levels in

Harvest and Stored Groundnut Kernels in Kaduna State, Nigeria. Journal of Environmental

Science, Toxicology and Food Technology. 9 (1): 62-66.

Wild, C.P and Turner, P.C. 2002. The toxicology of aflatoxins as a basis for public health decisions.

Mutagenesis. 17 (6): 471-481.

Williams, P and Norris, K. 2001. Near infrared technology in the agricultural and food industries.

American Association of Cereal Chemists.

Wu, F., 2004. Mycotoxin risk assessment for the purpose of setting international standards.

Environmental Science and Technology. 38: 4049-4055.

Wu, L.X., Ding, X.X., Li, P.W., Du, X.H., Zhou, H.Y., Bai, Y.Zh and Zhang, L.X. 2016. Aflatoxin

contamination of peanuts at harvest in China from 2010 to 2013 and its relationship with

climatic conditions. Food Control. 60: 117-123.

www.indiastat.com 2013.

Yitayih, G., Ayalew, A and Dechassa, N. 2013. Management of Aflatoxigenic fungi in groundnut

production in Eastern Ethiopia. East African Journal of Sciences. 7 (2): 85-98.

Yu, J., Chang, P.K., Ehrlich, K.C., Cary, J.W and Bhatnagar, D. 2004. Clustered pathway genes in

aflatoxin biosynthesis. Applied Environmental Microbiology. 70: 1253-1262.

Zain, M.E. 2011. Impact of mycotoxins on humans and animals. Journal of Saudi Chemical

Society. 15: 129-144.

http://www.indiastat.com/

EFFECT OF Aspergillus flavus ON GROUNDNUT SEED QUALITY …oar.icrisat.org/9725/1/Adithya.pdf · groundnut resistant cv. J 11 4.6 Scanning electron micrographs of A. flavus growth

Documents